General Laboratory Procedures

General Laboratory Procedures, Equipment Use, and
Safety Considerations
Arturo Duarte Ortuño
I. Safety Procedures
A. Chemicals
A number of chemicals used in any molecular biology laboratory are

hazardous. All manufacturers of hazardous materials are required by law
to supply the user with pertinent information on any hazards associated
with their chemicals. This information is supplied in the form of Material
Safety Data Sheets or MSDS. This information contains the chemical name,
CAS#, health hazard data, including first aid treatment, physical data, fire
and explosion hazard data, reactivity data, spill or leak procedures, and
any special precautions needed when handling this chemical. A file
containing MSDS information on the hazardous substances should be kept
in the lab. In addition, MSDS information can be accessed on World Wide
Web. You are strongly urged to make use of this information prior to using
a new chemical and certainly in the case of any accidental exposure or
spill. The instructor/lab manager must be notified immediately in the case
of an accident involving any potentially hazardous reagents.
The following chemicals are particularly noteworthy:
Phenol - can cause severe burns

Acrylamide - potential neurotoxin
Ethidium bromide - carcinogen
These chemicals are not harmful if used properly: always wear gloves
when using potentially hazardous chemicals and never mouth-pipet them.
If you accidentally splash any of these chemicals on your skin, immediately
rinse the area thoroughly with water and inform the instructor. Discard the
waste in appropriate containers.
B. Ultraviolet Light
Exposure to ultraviolet light can cause acute eye irritation. Since the retina
cannot detect UV light, you can have serious eye damage and not realize it
until 30 min to 24 hours after exposure. Therefore, always wear
appropriate eye protection when using UV lamps.
C. Electricity
The voltages used for electrophoresis are sufficient to cause electrocution.

Cover the buffer reservoirs during electrophoresis. Always turn off the
power supply and unplug the leads before removing a gel.
D. General Housekeeping
All common areas should be kept free of clutter and all dirty dishes,
electrophoresis equipment, etc should be dealt with appropriately. Since
you have only a limited amount of space to call your own, it is to your
advantage to keep your own area clean. Since you will use common
facilities, all solutions and everything stored in an incubator, refrigerator,
etc. must be labeled. In order to limit confusion, each person should use
his initials or other unique designation for labeling plates, etc. Unlabeled
material found in the refrigerators, incubators, or freezers may be
destroyed. Always mark the backs of the plates with your initials, the date,
and relevant experimental data, e.g. strain numbers.

II. Preparation of Solutions
A. Calculation of Molar, % and "X" Solutions .
1. A molar solution is one in which 1 liter of solution contains the number

of grams equal to its molecular weight. Ex. To make up 100 ml of a 5M
NaCl solution = 58.456 (mw of NaCl) g/mol x 5 moles/liter x 0.1 liter =
29.29 g in 100 ml of solution
2. Percent solutions.Percentage (w/v) = weight (g) in 100 ml of solution;

Percentage (v/v) = volume (ml) in 100 ml of solution. Ex. To make a 0.7%
solution of agarose in TBE buffer, weight 0.7 of agarose and bring up
volume to 100 ml with TBE buffer.
3. "X" Solutions. Many enzyme buffers are prepared as concentrated

solutions, e.g. 5X or 10X (five or ten times the concentration of the
working solution) and are then diluted such that the final concentration of
the buffer in the reaction is 1X. Ex. To set up a restriction digestion in 25 μ
l, one would add 2.5 μ l of a 10X buffer, the other reaction components,
and water to a final volume of 25 μ l.
B. Preparation of Working Solutions from Concentrated Stock Solutions .
Many buffers in molecular biology require the same components but often
in varying concentrations. To avoid having to make every buffer from
scratch, it is useful to prepare several concentrated stock solutions and
dilute as needed. Ex. To make 100 ml of TE buffer (10 mM Tris, 1 mM
EDTA), combine 1 ml of a 1 M Tris solution and 0.2 ml of 0.5 M EDTA and
98.8 ml sterile water. The following is useful for calculating amounts of
stock solution needed: C i x V i = C f x V f , where C i = initial
concentration, or conc of stock solution; V i = initial vol, or amount of stock
solution needed C f = final concentration, or conc of desired solution; V f =
final vol, or volume of desired solution
C. Steps in Solution Preparation:
Refer to a laboratory reference manual for any specific instructions on

preparation of the particular solution and the bottle label for any
specific precautions in handling the chemical. Weigh out the
desired amount of chemical(s). Use an analytical balance if the
amount is less than 0.1 g. Place chemical(s) into appropriate size
beaker with a stir bar. Add less than the required amount of water.
Prepare all solutions with double distilled water When the chemical
is dissolved, transfer to a graduated cylinder and add the required
amount of distilled water to achieve the final volume. An exception
is in preparing solutions containing agar or agarose. Weigh the
agar or agarose directly into the final vessel. If the solution needs
to be at a specific pH, check the pH meter with fresh buffer
solutions and follow instructions for using a pH meter. Autoclave, if
possible, at 121 deg C for 20 min. Some solutions cannot be
autoclaved, for example, SDS. These should be filter sterilized
through a 0.22 μ m or 0.45 μ m filter. Media for bacterial cultures
must be autoclaved the same day it is prepared, preferably within
an hour or two. Store at room temperature and check for
contamination prior to use by holding the bottle at eye level and
gently swirling it Solid media for bacterial plates can be prepared
in advance, autoclaved, and stored in a bottle. When needed, the
agar can be melted in a microwave, any additional components,
e.g. antibiotics, can be added and the plates can then be poured.
Concentrated solutions, e.g. 1M Tris-HCl pH=8.0, 5M NaCl, can be used to
make working stocks by adding autoclaved double-distilled water
in a sterile vessel to the appropriate amount of the concentrated
solution.
D. Glassware and Plastic Ware .
Glass and plastic ware used for molecular biology must be scrupulously
clean. Dirty test tubes, bacterial contamination and traces of detergent can
inhibit reactions or degrade nucleic acid.
Glassware should be rinsed with distilled water and autoclaved or baked at

150 degrees C for 1 hour. For experiments with RNA, glassware and
solutions are treated with diethyl-pyrocarbonate to inhibit RNases which
can be resistant to autoclaving. Plastic ware such as pipets and culture
tubes are often supplied sterile. Tubes made of polypropylene are turbid
and are resistant to many chemicals, like phenol and chloroform;
polycarbonate or polystyrene tubes are clear and not resistant to many
chemicals. Make sure that the tubes you are using are resistant to the
chemicals used in your experiment. Micro pipet tips and microfuge tubes
should be autoclaved before use.
III. Methods for DNA isolation

A. Large scale double-stranded DNA isolation
The method used for the isolation of large scale cosmid and plasmid DNA is an
unpublished modification (16) of an alkaline lysis procedure (17,18) followed by
equilibrium ultracentrifugation in cesium chloride-ethidium bromide gradients (1).
Briefly, cells containing the desired plasmid or cosmid are harvested by centrifugation,
incubated in a lysozyme buffer, and treated with alkaline detergent. Detergent
solubilized proteins and membranes are precipitated with sodium acetate, and the lysate
is cleared first by filtration of precipitate through cheesecloth and then by
centrifugation. The DNA-containing supernatant is transferred to a new tube, and the
plasmid or cosmid DNA is precipitated by the addition of polyethylene glycol and
collected by centrifugation. The DNA pellet is resuspended in a buffer containing
cesium chloride and ethidium bromide, which is loaded into polyallomer tubes and
subjected to ultracentrifugation overnight. The ethidium bromide stained plasmid or
cosmid DNA bands, equilibrated within the cesium chloride density gradient after
ultracentrifugation, are visualized under long wave UV light and the lower band is
removed with a 5 cc syringe. The intercalating ethidium bromide is separated from the
DNA by loading the solution onto an equilibrated ion exchange column. The A260
containing fractions are pooled, diluted, and ethanol precipitated, and the final DNA
pellet is resuspended in buffer and assayed by restriction digestion as detected on
agarose gel electrophoresis.
During the course of this work several modifications to the above protocol were made.
For example, initially cell growth times included three successive overnight
incubations, beginning with the initial inoculation of 3 ml of antibiotic containing media
with the plasmid or cosmid-containing bacterial colony, and then increasing the culture
volume to 50 ml, and then to 4 l. However, it was observed that recombinant cosmid
DNA isolated from cell cultures grown under these conditions, in contrast to
recombinant plasmid DNA, was contaminated with deleted cosmid DNA molecules.
However, these deletions are avoided by performing each of the three successive
incubations for eight hours instead of overnight, although a slight yield loss
accompanied the reduced growth times.
Recently, a diatomaceous earth-based (19-22) method was used to isolate the plasmid or
cosmid DNA from a cell lysate. The cell growth, lysis, and cleared lysate steps are
performed as described above, but following DNA precipitation by polyethylene glycol,
the DNA pellet is resuspended in RNase buffer and treated with RNase A and T1.
Nuclease treatment is necessary to remove the RNA by digestion since RNA competes
with the DNA for binding to the diatomaceous earth. After RNase treatment, the DNA
containing supernatant is bound to diatomaceous earth in a chaotropic buffer of
guanidine hydrochloride by incubation at room temperature. The DNA-associated
diatomaceous earth then is collected by centrifugation, washed several times with
ethanol buffer and acetone, dried, and then resuspended in buffer. The DNA is eluted
during incubation at 65degC, and the DNA-containing supernatant is collected after
centrifugation and separation of the diatomaceous earth particles. The DNA recovery is
measured by taking absorbance readings at 260 nanometers. After concentration by
ethanol precipitation, the DNA is assayed by restriction digestion.
Protocol
1. Pick a colony of bacteria harboring the plasmid or cosmid DNA of interest into a 12
X 75 mm Falcon tube containing 2 ml of LB media supplemented with the appropriate
antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg C 8-10 hours with
shaking at 250 rpm. Transfer the culture to an Ehrlenmeyer flask containing 50 ml of
similar media, and incubate further for 8-10 hours. Transfer 12.5 ml of the culture to
each of 4 liters of similar media, and incubate for an additional 8-10 hours.
2. Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml bottles in
the RC5-B using the GS3 rotor. Resuspend the cell pellets in old media and transfer to
two bottles, centrifuge as before, and decant the media. The cell pellets can be frozen at
-70degC at this point.
3. Resuspend the cell pellets in a total of 70 ml of GET/Lysozyme solution (35 ml for

each bottle) by gently teasing the pellet with a spatula and incubate for 10 minutes at
room temperature. (Note: Do not vortex the lysate at any time because this may shear
the chromosomal DNA).
4. Add a total of 140 ml of alkaline lysis solution (70 ml for each bottle), gently mix,
and incubate for 5 minutes in an ice-water bath.
5. Add 105 ml of 3M NaOAc, pH 4.8 (52.5 ml for each bottle), cap tightly, gently mix
by inverting the bottle a few times, and incubate in an ice-water bath for 30-60 minutes.
6. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal DNA
by pouring through a double-layer of cheesecloth. Transfer the lysate into 250 ml
centrifuge bottle, centrifuge at 10,000 rpm for 30 minutes at 4deg C in the RC5-B using
the GSA rotor.
For cesium chloride-gradient purification:
7a. Pool the cleared supernatants into to a clean beaker, add one-fourth volume of 50%
PEG/0.5 M NaCl, swirl to mix, and incubate in an ice-water bath for 1-2 hours.
8a. Collect the PEG-precipitated DNA by centrifugation in 250 ml bottles at 7000 rpm
for 20 minutes at 4degC in the RC5-B using the GSA rotor.
9a. Dissolve the pellets in a combined total of 32 ml of 100:10 TE buffer, 5 ml of 5

mg/ml ethidium bromide, and 37 g cesium chloride (Var Lac Oid Chemical Co., Inc.)
(final concentration of cesium chloride should be 1 g/ml).
10a. Transfer the sample into 35 ml polyallomer centrifuge tubes, remove air bubbles,
seal with rubber stoppers, and crimp properly.
11a. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in the Sorvall OTD-75B
ultracentrifuge (DuPont) using the T-865 rotor.
12a. Visualize the ethidium bromide stained DNA under long-wave UV light, and
remove the lower DNA band using a 5 cc syringe and a 25 gauge needle. (It may be
helpful first to remove and discard the upper band).
13a. To remove the ethidium bromide, load the DNA sample onto an equilibrated 1.5 ml
Dowex column, and collect 0.5 ml fractions. Equilibrate the Dowex AG resin (BioRad)
by successive centrifugation, resuspension, and decanting with 1M NaOH, water, and
then 1M Tris-HCl, pH 7.6 until the Dowex solution has a pH of 7.6.
14a. Pool fractions with an A260 of 1.00 or greater into 35 ml Corex glass tubes, add
one volume of ddH2O, and ethanol precipitate by adding 2.5 volumes of cold 95%
ethanol. Incubate at least 2 hours at -20degC, centrifuge at 10,000 rpm for 45 minutes in
the RC5-B using the SS-34 rotor. Gently decant the supernatant, add 80% ethanol,
centrifuge as before, decant, and dry the DNA pellet in a vacuum oven.
15a. Resuspend the DNA in 10:0.1 TE buffer.
For diatomaceous earth-based purification:
7b. Pool the supernatants from step 6 into 500 ml bottles and add DNase-free RNase A
and RNase T1 such that the final concentration of RNase A is 40 ug/ml and RNase T1 is
40 U/ml. Incubate in a 37degC water bath for 30 minutes.
8b. Add an equal volume of isopropanol and precipitate at room temperature for 5
minutes. Centrifuge at 9,000 rpm for 30 minutes in the RC5-B using the GS3 rotor.
Decant the supernatant and drain the DNA pellet.
9b. Resuspend each DNA pellet in 20 ml 10:1 TE buffer, and add 40 ml of de-fined
diatomaceous earth in guanidine-HCl (100 mg/ml) to each bottle. Allow the DNA to
bind at room temperature for 5 minutes with occasional mixing. Centrifuge at 9,000 for
10 minutes in the RC5-B using the GS3 rotor.
10b. Decant the supernatant, resuspend each pellet in 40 ml of diatomaceous earth-wash

buffer, and centrifuge as above.
11b. Decant the supernatant, resuspend each pellet in 40 ml of acetone, and centrifuge
as above.
12b. Decant the supernatant and dry the pellet in a vacuum oven.
13b. Resuspend the pellet in 20 ml of 10:1 TE buffer, and elute the bound DNA by
incubation at 65degC for 10 minutes with intermittent mixing.
14b. Remove the diatomaceous earth by centrifugation at 9,000 rpm for 10 minutes in
the RC5-B using the GS3 rotor. Repeat if necessary.
15b. Combine the DNA-containing supernatants and precipitate the DNA in 35 ml

Corex glass tubes adding 2.5 volumes of cold 95% ethanol/acetate.
16b. Resuspend the dried DNA pellet in 2 ml of 10:0.1 TE buffer and assay for
concentration by absorbance readings at 260 nm or by agarose gel electrophoresis.
B. Midiprep double-stranded DNA isolation

A midi-prep double-stranded DNA isolation has been developed to generate a sufficient
amount of template DNA for several Sequenase[TM] catalyzed fluorescent terminator
reactions. Here, one bacterial colony which harbored the plasmid of interest is picked
into 3 ml of liquid media containing ampicillin and incubated in a 37degC shaker for 8-
10 hours. At this time, the culture is transferred into 50 ml of ampicillin-containing
media and incubated further for 10-12 hours. After harvesting the cells by
centrifugation, a diatomaceous earth-based alkaline-lysis purification method (19-22) is
performed, similar to that discussed above for large scale DNA isolation. The purified
DNA is crudely assayed for concentration and purity by agarose gel electrophoresis
against known standards. The approximate yield of double-stranded DNA using this
method is 1 ug of DNA per ml of cell culture. For a 50 ml cell culture, about 50 ug of
DNA are recovered, and 5 ug are used typically in a Sequenase[TM] terminator
reaction.
Note: This procedure is the method of choice for isolating double stranded plasmid-
based templates for the Sequenase Dye-Labeled Terminator Sequencing Reactions.
Protocol
1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 12 X 75 mm

Falcon tube containing 3 ml of 2xTY media supplemented with the appropriate
antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg C 8-10 hours with
shaking at 250 rpm. Transfer the culture to an Ehrlenmeyer flask containing 50 ml of
similar media, and incubate further for 11-14 hours.
2. Harvest the cells by centrifugation at 3000 rpm for 5 minutes in 50 ml conical tubes
in the Beckman GPR tabletop centrifuge and decant the supernatant. The cell pellets can
be frozen at -70degC at this point.
3. Resuspend the cell pellets in 2 ml of GET/Lysozyme solution, add 4 ml of alkaline

lysis solution, gently mix, and incubate for 5 minutes in an ice-water bath.
4. Add 4 ml of 3M NaOAc, pH 4.8, gently mix by swirling, and incubate in an ice-water

bath for 30-60 minutes.
by pouring through a double-layer of cheesecloth into a new 50 ml conical tube.
Centrifuge at 3,000 rpm for 20 minutes at 4degC in the Beckman GPR tabletop
centrifuge.
6. Decant the supernatant to a 50 ml polypropylene centrifuge tube, add 20 ul of a 20

mg/ml DNase-free RNase A and incubate in a 37degC water bath for 30 minutes.
7. Add 7 ml (equal volume) of de-fined diatomaceous earth in guanidine-HCl (20

mg/ml) and allow the DNA to bind at room temperature for 5 minutes with occasional
mixing. Centrifuge at 3,000 for 5 minutes in the Beckman GPR tabletop centrifuge.
8. Decant the supernatant, resuspend in 7 ml of diatomaceous earth-wash buffer, and

centrifuge as above.
9. Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as above.
10. Decant the supernatant and dry in a vacuum oven.
11. Resuspend the pellet in 0.6 ml of 10:1 TE buffer, and elute the bound DNA by
incubation at 65degC for 10 minutes with intermittent mixing.
12. Remove the diatomaceous earth by centrifugation at 3,000 rpm for 5 minutes in the
in the Beckman GPR tabletop centrifuge.
13. Transfer the supernatant to a 1.5 ml microcentrifuge tube and centrifuge at 12,000
rpm for 5 minutes in a microcentrifuge at room temperature. Transfer the supernatant to
a new 1.5 ml microcentrifuge tube and ethanol precipitate.
14. Resuspend the dried DNA pellet in 40 ul of 10:0.1 TE buffer and assay for
concentration by agarose gel electrophoresis.
C. Miniprep double-stranded DNA isolation

The standard method for the miniprep isolation of plasmid DNA includes the same
general strategy as the large scale isolation. However, smaller aliquots of antibiotic
containing liquid media inoculated with plasmid-containing cell colonies are incubated
in a 37degC shaker for 12-16 hours. After collecting the plasmid containing cells by
centrifugation, the cell pellet is resuspended in a hypotonic sucrose buffer. The cells are
successively incubated with an RNase-lysis buffer, alkaline detergent, and sodium
acetate. The lysate is cleared of precipitated proteins and membranes by centrifugation,
and the plasmid DNA is recovered from the supernatant by isopropanol precipitation.
The DNA is crudely checked for concentration and purity using agarose gel
electrophoresis against known standards. A typical yield for this method of DNA
isolation is 10-15 ug of plasmid DNA from a 6 ml starting culture.
Since highly supercoiled DNA is desired for double-stranded DNA sequencing, a

modification of this method employing diatomaceous earth (19-22) sometimes is used
for isolation of double-stranded templates for DNA sequencing with fluorescent
primers. After removal of the precipitated proteins and membranes, the plasmid
containing supernatant is incubated with diatomaceous earth and guanidine
hydrochloride and this mixture is added into one of the twenty-four wells in the BioRad
Gene Prep Manifold. The supernatant is removed by vacuum filtration over a
nitrocellulose filter. The DNA-associated diatomaceous earth is washed to remove the
guanidine hydrochloride with an ethanol buffer, and then dried by filtration. Elution
buffer is added to the wells, and the DNA-containing solution then is separated from the
diatomaceous earth particle by filtration into a collection tube. The collected DNA is
concentrated by ethanol precipitation and crudely assayed for concentration and purity
by agarose gel electrophoresis against known standards. The approximate yield of
double-stranded DNA is 3-5 ug of DNA from 6 ml of starting culture.
Note: This is a typical mini-prep until step 7, where in step 7a you would precipitate the
template and use it for Taq Cycle Sequencing with the Dye-Labeled Primers, or in step
7b proceed with the diatomaceous earth purification for Taq Dye-Labeled Terminator
Cycle Sequencing Reactions. For Sequenase Dye-Labeled Terminator Sequencing
Reactions use the Midi-prep procedure detailed above.
Protocol
1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 17 X 100 mm
Falcon tube containing 6 ml of TB media supplemented with the appropriate antibiotic
(typically ampicillin at 100 ug/ml) and incubate at 37deg C 16-18 hours with shaking at
250 rpm.
2. Harvest the cells by centrifugation at 3000 rpm for 5 minutes in the Beckman GPR
tabletop centrifuge and decant the supernatant. The cell pellets can be frozen at -70degC
at this point.
3. Resuspend the cell pellets in 0.2 ml of TE-RNase solution (50:10 TE buffer

containing 40 ug/ml RNase A; some also add RNase T1 to a final concentration of 10
U/ul) by gentle vortexing, add 0.2 ml of alkaline lysis solution, gently mix, and incubate
for 15 minutes at room temperature.
4. Add 0.2 ml of 3M NaOAc, pH 4.8, gently mix by swirling, transfer to 1.5 ml

microcentrifuge tubes, and incubate in an ice-water bath for 15 minutes.
by centrifugation at 12,000 rpm for 15 minutes in a microcentrifuge at 4deg C.
6. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube, incubate in an ice-

water bath for 15 minutes, centrifuge as above for an additional 15 minutes and transfer
the supernatant to a clean 1.5 ml tube.
For standard alkaline lysis purification:
7a. Precipitate the DNA by adding 1 ml of 95% ethanol, and resuspend the dried DNA
pellet in 100-200 ul 10:0.1 TE buffer. Electrophorese an aliquot of the DNA sample on
a 0.7% agarose gel to crudely determine the concentration and purity.
For diatomaceous earth-base purification:
7b. Add 1 ml of de-fined diatomaceous earth in guanidine-HCl (20 mg/ml) and allow
the DNA to bind at room temperature for 5 minutes with occasional mixing. Meanwhile
soak the Prep-A-Gene nitrocellulose membrane in isopropanol for at least 3 minutes,
and assemble the Prep-A-Gene manifold as described in the manual.
8b. Turn on the vacuum pump and adjust the vacuum level to 8 in. Hg, let the
membrane dry for 1 minute, and then release the vacuum.
9b. Pour the well mixed samples into the wells of the Prep-A-Gene manifold and filter
through at 8 in. Hg until all the liquid is filtered through.
10b. Wash the samples four times with 250 ul of diatomaceous earth-wash buffer, using
a repeat pipette, allowing all of the liquid to filter through between washes.
11b. Reduce the vacuum to 5 in. Hg before turning the vacuum off at the stopcock.
Without unscrewing the black clamps, release the white clamps and place the collection
rack with clean 1.5 ml screw-capped tubes into the manifold. Clamp the manifold with
the white clamps, and apply 300 ul of 10:1 TE buffer heated to 65degC and pull the
eluted DNA through at 5 in. Hg. After the liquid has filtered through, raise the vacuum
to 10-12 in. Hg, and let the membrane dry for 1 minute.
12b. Turn off the vacuum at the stopcock and remove the collection rack containing the
tubes. Ethanol precipitate the DNA and resuspend the dried DNA pellet in 30 ul of
10:0.1 TE buffer.
D. Large scale M13RF isolation (9)

Double-stranded M13RF is isolated for use in M13 SmaI cut, dephosphorylated vector
preparation, described below. The growth conditions of M13-infected bacterial cells
(see Figure 1) appears convoluted, but result in a maximal amount of M13 RF
molecules per cell. After the M13RF containing bacterial cells are harvested by
centrifugation, the double-stranded molecules are isolated using the cesium chloride
method for large scale plasmid isolation, as described above. This briefly entailed
alkaline cell lysis, sodium acetate precipitation of detergent solubilized proteins and
membranes, polyethylene glycol DNA precipitation, and extraction of ethidium bromide
stained DNA from a cesium chloride gradient after ultracentrifugation. After removal of
the ethidium bromide on an ion-exchange column, the DNA containing fractions are
detected by A260 measurement and pooled, and the DNA is concentrated by ethanol
precipitation and assayed by restriction enzyme digestion and agarose gel
electrophoresis.
Protocol
1. Prepare an early log phase culture of JM101 by inoculating an Ehrlenmeyer flask

containing 50 ml of 2xTY with a glycerol stock of JM101 and pre-incubating for 1 hour
in a 37degC water bath, with no shaking. Pick a plaque representing the desired M13
clone into four 1.5 ml aliquot of early log phase JM101, and incubate according to the
procedure displayed in Figure 1 to result in 4 liters of M13-infected bacteria.
2. Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml bottles in
the RC5-B using the GS3 rotor. Resuspend the cell pellets in fresh 2xTY media to
remove contaminating extracellular phage and transfer to two bottles, centrifuge as
before, and decant the media. The cell pellets can be frozen at -70degC at this point.
3. Resuspend the cell pellets in a total of 120 ml (30 ml for each bottle) of 1X STB
buffer by gently teasing the pellet with a spatula. Add a total 24 ml of lysozyme solution
(6 ml for each bottle), gently mix, and incubate for 5 minutes in an ice-water bath.
4. Add 48 ml of 50:2:10 TTE buffer (12 ml for each bottle) and 2 ml of RNase A (10
mg/ml) (0.5 ml for each bottle), gently mix, and incubate in an ice-water bath for 5
minutes.
by pouring through a double-layer of cheesecloth. Transfer the lysate into 250 ml
centrifuge bottle, centrifuge at 10,000 rpm for 30 minutes at 4deg C in the RC5-B using
the GSA rotor.
6. Add 6 ml of 5 mg/ml ethidium bromide, and cesium chloride such that the final
concentration of cesium chloride is 1 g/ml.
7. Transfer the sample into 35 ml polyallomer centrifuge tubes and top off with a 1:1
solution of 100:10 TE buffer and cesium chloride, remove air bubbles, seal with rubber
stoppers, and crimp properly.
8. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in the Sorvall OTD-75B

ultracentrifuge using the T-865 rotor.
9. Visualize the ethidium bromide stained DNA under long-wave UV light, and remove
the lower DNA band using a 5 cc syringe and a 25 gauge needle. (It may be helpful to
remove and discard the upper band first).
10. To remove the ethidium bromide, load the DNA sample onto an 1.5 ml Dowex AG
(BioRad) column, equilibrated as before, and collect 0.5 ml fractions.
11. Pool fractions with an A260 of 1.00 or greater into 35 ml Corex glass tubes, add one
volume of ddH2O, and ethanol precipitate by adding 2.5 volumes of cold 95% ethanol.
Incubate at least 2 hours at -20degC, centrifuge at 10,000 rpm for 45 minutes in the
RC5-B using the SS-34 rotor. Gently decant the supernatant, add 80% ethanol,
centrifuge as before, decant, and dry the DNA pellet in a vacuum oven.
12. Resuspend the DNA in 10:0.1 TE buffer.
E. Single-stranded M13 DNA isolation using phenol

This isolation procedure (23) is the method of choice for preparation of M13-based
templates to be used in Sequenase[TM] catalyzed dye-terminator reactions. A pre-
incubated early log phase JM101 culture is prepared by transferring a thawed glycerol
stock into 50 ml of liquid media and incubating for 1 hour at 37degC with no shaking.
M13 plaques are picked with a sterile toothpick and placed into 1.5 ml aliquots of the
early log phase JM101 culture, which are incubated in a 37deg C shaker for 4-6 hours.
After incubation, the bacterial cells are pelleted by centrifugation and the viral
containing supernatant is transferred to a clean tube. The phage particle are precipitated
with PEG, collected by centrifugation, and the pellet is resuspended in buffer. The
phage protein coat is denatured and removed by one phenol and two ether extractions.
After ethanol precipitation, the dried DNA pellet is resuspended in buffer, and the
concentration and purity crudely are assessed by agarose gel electrophoresis against
known standards.
Protocol
1. Prepare an early log phase culture of JM101, as above, and pick M13-based plaques
with sterile toothpicks into 12 X 75 mm Falcon tubes containing 1.5 ml aliquots of the
cells. Incubate for 4-6 hours at 37degC with shaking at 250 rpm.
2. Transfer the culture to 1.5 ml microcentrifuge tubes and centrifuge for 15 minutes at
12,000 rpm at 4degC.
3. Pipette the top 1 ml of supernatant to a fresh 1.5 ml microcentrifuge tube containing

0.2 ml 20% PEG/2.5 M NaCl to precipitate the phage particles. Mix by inverting several
times and incubate for 15-30 minutes at room temperature.
4. Centrifuge for 15 minutes at 12,000 rpm at 4degC to collect the precipitated phage.
Decant the supernatant and remove residual PEG supernatant by suctioning twice.
5. Resuspend the pellet in 100 ul of 10 mM Tris-HCl, pH 7.6 by vortexing, and add 50

ul of TE-saturated phenol.
6. Extract the DNA with phenol and twice with ether, as discussed above, and then
ethanol precipitate.
7. Resuspend the dried DNA in 6 ul of 10:0.1 TE for use in single-stranded

Sequenase[TM] catalyzed dye-terminator sequencing reactions.
F. Biomek-automated modified-Eperon isolation

procedure for single-stranded M13 DNA
This semi-automated method is a modification of a previously reported procedure
(24,25), and allowed the simultaneous isolation of 48 single-stranded DNAs per Biomek
1000 robotic workstation within 3 hours (26). Basically, M13 plaques are picked with
sterile toothpicks into aliquots of early log phase JM101, prepared as discussed above.
The phage infected cultures are incubated in a 37degC shaker for 4-6 hours, transferred
into microcentrifuge tubes, centrifuged to separate bacterial cells from the viral
supernatant, and then carefully placed on the Biomek tablet. For each sample, two 250
ul aliquots are robotically distributed into two wells of a 96-well microtiter plate, and
this process is repeated for each of the 48 samples until the entire 96 wells are filled. A
solution of polyethylene glycol (PEG) then is added robotically to each well and mixed.
The microtiter plate is covered with an acetate plate sealer, incubated at room
temperature to precipitated the phage particles, and then centrifuged. The supernatant
then is removed by inverting the plate and gently draining on a paper towel, without
dislodging the pellet. After placing the microtiter plate back on the Biomek, a more
dilute PEG solution is robotically added to each well. The plate then is covered with
another sealer and centrifuged again. This rinse step aided in the removal of
contaminating proteins and RNA. After removing the supernatant, as before, and
placing the microtiter plate back on the Biomek, a Triton X-100 detergent solution is
robotically added to each well. The plate is agitated gently and the sample from each
pair of wells is robotically transferred to microcentrifuge tubes, which then are capped
and placed in an 80deg C water bath for 10 minutes to aid in the detergent solubilization
of phage coat proteins. After a brief centrifugation to collect condensation, the single-
stranded DNA is ethanol precipitated, dried, and resuspended. An aliquot from each
DNA sample is subjected to agarose gel electrophoresis to crudely assay concentration
and purity. The yield of single-stranded template is approximately 2-3 ug per sample.
Protocol
The entire procedure will require 9 rows of P250 tips (counting from the center of the
Biomek tablet towards the left) for the isolation of 48 templates (48ISOL). The reagent
module should contain PEG-2000, Triton-Tris-EDTA, and ethanol-acetate, respectively.
1. Prepare an early log phase JM101 culture in 50 ml of 2xTY, as above.
2. Using sterile toothpicks, transfer individual M13 plaques into 12 X 75 mm Falcon

tubes containing 1 ml early log phase cell cultures, and incubate for 4-6 hours at
37degC with shaking at 250 rpm. (Growth for longer than 6 hours results in cell lysis
and contamination of the phage DNA by cellular proteins and nucleic acids).
3. Separate the bacterial cells from the viral-containing supernatant by centrifugation at

12,000 rpm for 15 minutes at 4degC. Carefully open the tubes and place on the Biomek
tablet..
4. The Biomek will distribute two 250 ul aliquots of viral supernatant per sample into
the wells of a 96-well flat-bottomed microtiter plate (Dynatech). The Biomek then will
add 50 ul of 20% PEG/2.5 M NaCl solution to each well, and mix by pipetting up and
down.
5. Cover the plate with an acetate plate sealer and incubate at room temperature for 15
minutes.
6. Pellet the precipitated phage by centrifuging the plate at 2400 rpm for 20 minutes in a
Beckman GPR tabletop centrifuge. Remove the plate sealer and drain the PEG from the
plate by gently draining upside down on a Kimwipe.
7. Return the plate to the tablet, and the Biomek will robotically add 200 ul of PEG:TE
rinse solution to each well. Cover the plate with a plate sealer, centrifuge, and drain, as
above.
8. Return the plate to the tablet, and the Biomek will add 70 ul of TTE solution to each
well. Remove and gently agitate to resuspend.
9. The Biomek then will robotically pool the contents from each pair of wells into 1.5
ml microcentrifuge tubes.
10. Incubate the tubes at 80degC for 10 minutes to denature the viral protein coat and
then centrifuge briefly to reclaim condensation.
11. Ethanol precipitate the DNA by adding 500 ul ethanol/acetate to each tube, as
described above.
12. Resuspend the DNA templates in 20 ul of 10:0.1 TE buffer.
G. 96 well double-stranded template isolation

A manual as well as an automated procedure is given below. The automated method is a
modification of a previously reported procedure (4) which allows simultaneous isolation
of 96 double stranded DNAs per Biomek 1000 Automated Laboratory Workstation
within two hours. Basically colonies containing double-stranded plasmids are picked
with sterile toothpicks into media and incubated at 37degC for 24 hours with shaking at
350 rpm. These cells are harvested by centrifugation and the pellets are either manually
or robotically resuspended by the addition of TE-RNase solution. An alkaline lysis
solution is used to lyse the cells and the lysate is precipitated with KOAc. The lysate is
cleared by filtration and further concentrated by ethanol precipitation. An aliquot from
each DNA sample is subjected to agarose gel eletrophoresis to crudely assay
concentration and purity. The yield of double stranded template is approximately 3 mg
per sample.
Protocol
Manual Double stranded isolation method
The following is a manual, 96 well, double stranded sequencing template isolation

procedure that has been developed in our laboratory. A similar procedure that has been
automated on the Biomek is presented elsewhere herein.
1. Pick individual shotgun clones off a plate with a steril tooth pick and deposit each
separately into 96 well block containing 1.75 ml of TB media per well. Keep toothpick
in media for about 5 minutes to allow the cells to defuse into the media, remove the
toothpicks, cover the 96 well block with the loose fitting lid, and allow the cells to grow
for 24 hours in the 37degC shaker/incubator at 350 rpm.
2. Remove block from the shaker/incubator and collect the cells by centrifugation at
2500 rpm for 7 minutes. The cells can be stored frozen at -20degC in the block at this
stage.
3. After thawing the cells, add 100ul TE-RNAse-A solution containing RNAse T1, mix
by pipetting up and down 4-5 times to resuspend the cell pellet and then incubate in the
37degC incubator/shaker for 5 minutes at 350 rpm to mix more thoroughly.
4. Remove the block from the incubator/shaker and then add 100ul of alkaline lysis
solution. Shake the block by hand to mix the reagents and then incubate at room
temperature for 1 hour with intermittent swirling. 5. Then add 100ul of either 3M
potassium or sodium acetate, pH 5, and place the block in the 37degC shaker/incubator
for 5 minutes at 350 rpm to thoroughly mix and shear genomic DNA to reduce the
viscosity of the solution. Place the block at -20degC for 30 minutes.
6. Centrifuge the block in the GPR centrifuge at 3000 rpm at 4degC for 30 minutes.
7. Carefully remove 200 ul of the supernatant from each well in the 96 well block with
the 12 channel pipetter and transfer them to a v-bottom microtiter plate, being careful
not to transfer any cell debris.
8. Transfer 10 ul of supernatant into the respective cycle sequencing reaction tubes, and
precipitate with 150 ul of 95% ethanol (without added acetate). After storage at
-20degC for 30 minutes, the pellet is collected by centrifugation, washed three times
with 70% ethanol, and dried directly in the cycle sequencing reaction tubes.
9. Prior to adding the fluorscent terminator cycle sequencing reaction mix, the dried
templates should be stored at -20degC. An additional 75 ul of the supernatant is
transfered to a Robbins PCR reaction tube (in 96 well tube format) and precipitated with
200 ul of 95% ethanol, washed three times with 70% ethanol, and stored dry at -20degC
for future use.
The following is an automated, 96 well, double stranded sequencing template isolation

procedure that has been developed in our laboratory.
1. Pick colonies using a toothpick into 1.8 ml TB with TB salts containing appropriate
antibiotic and shake for 22-24 hours at 350 rpm in a 96 well block with cover.
2. Harvest cells by centrifugation at 1800 rpm for 7 min. Pour off supernatant and allow
pellets to drain inverted. Cell pellets may be frozen at this point if necessary.
3. Turn on Biomek, begin the program DSISOL2 and set up the Biomek as indicated in
the configuration function on the screen. Specifically, you should put TE-RNase
solution in the first module, alkaline lysis solution in the second reagent module and 3
M KOAc, pH 4.8 in the third module.
4. Place the 96 well block containing cells onto the Biomek tablet at the position labeled
"1.0 ml Minitubes". Place a Millipore filter plate in the position labeled "96well flat
bottomed microtitre plate".
5. Press ENTER to continue with the program.
6. First the Biomek will add 100 ml TE-RNase solution to the cell pellets and mix to
partially resuspend.
7. Next, the biomek will add 100 ml alkaline lysis solution to the wells of the filter
plate.
8. The biomek then will mix the cell suspension again, transfer the entire volume to the
filter plate containing alkaline lysis solution, and mix again. Set up the filtration
apparatus with a clean 96 well block to collect the filtrate (wash and reuse the block
used for growth).
9. The biomek will add 100 ml 3M KOAc, pH 4.8 to the wells of the filter plate and mix
at the sides of the wells. Some choose to place the filter plate at -20degC for 5 minutes
at this point. Transfer the filter plate to the QiaVac Vacuum Manifold 96 and filter
using water vacuum only (do not do a harsh filtration as the plates are fragile and will
loose their seal). This will typically take less than 20 minutes.
10. The supernatant collected in the 96 well block is the crude DNA and must be
ethanol precipitated before use by the addition of 1 ml 100% ethanol and incubation at
-20degC for at least 30 minutes.
11. Centrifuge for 25 minutes at 3000 rpm in a cooled Beckman GPR centrifuge.
12. Decant and wash with 500 ml 80% ethanol and centrifuge for an additional 5
minutes at 3000 rpm.
13. Decant the supernatant, drain inverted on a paper towel. Dry under vacuum.
14. Resuspend in 50 ml 10:0.1 TE for use in dye primer or dye terminator sequencing
chemistry.
H. Genomic DNA isolation from blood

Genomic DNA isolation is performed according to the FBI protocol (27). After the
blood samples (stored at -70degC in EDTA vacutainer tubes ) are thawed, standard
citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the
supernatant is discarded and additional buffer is added, mixed, and again the tube is
centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of
SDS detergent and proteinase K, and the mixture is incubated at 55deg C for one hour.
The sample then is phenol extracted once with a phenol/chloroform/isoamyl alcohol
solution, and after centrifugation the aqueous layer is removed to a fresh
microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then
ethanol precipitated a second time. Once the pellet is dried, buffer is added and the
DNA is resuspended by incubation at 55degC overnight, the genomic DNA solution is
assayed by the polymerase chain reaction.
Protocol (updated 10/30/98)
1. Blood samples typically were obtained as 1 ml of whole blood stored in EDTA

vacutainer tubes frozen at -70deg C.
2. Thaw the frozen samples, and to each 1 ml sample, add 0.8 ml 1X SSC buffer, and
mix. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.
3. Remove 1 ml of the supernatant and discard into disinfectant.
4. Add 1 ml of 1X SSC buffer, vortex, centrifuge as above for 1 minute, and remove all
of the supernatant.
5. Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 ul of 10%
SDS and 5 ul of proteinase K (20 mg/ml H2O) (Sigma P-0390), vortex briefly and
incubate for 1 hour at 55degC.
6. Add 120 ul phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge

the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.
7. Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of
cold 100% ethanol, mix, and incubate for 15 minutes at -20deg C.
8. Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant

and drain.
9. Add 180 ul 10:1 TE buffer, vortex, and incubate at 55degC for 10 minutes.
10. Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and
centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.
11. Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for
1 minute at 12,000 rpm in a microcentrifuge.
12. Decant the supernatant, and dry the pellet in a Speedy-Vac for 10 minutes (or until
dry).
13. Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at
55degC, vortexing periodically to dissolve the genomic DNA. Store the samples at
-20degC.
Bruce A. Roe, Department of Chemistry and Biochemistry, The University of

Oklahoma, Norman, Oklahoma 73019 broe@ou.edu
DNA Extraction
This is a modification of a salting out procedure as described by Miller et al., (1988),
evaluated at the DNA Laboratory, Medical School, Malta. When analysed by
spectrophotometry >95% of extracted genomic DNA gave a purity ratio of DNA to
proteins in the range of 1.7 - 1.9 and a concentration of 100ng/ml.
In the following protocol DNA was extracted from peripheral blood leucocytes using
3mls of whole blood. It was observed that on prolonged storage of whole blood at -20
and -80ºC , DNA yield decreased considerably probably due to degeneration of the
white blood cells. Volumes of buffers and reagents used may be adjusted according to
volume of whole blood used.
PROCEDURE:
1. Whole blood was collected in disodium EDTA containers and stored at -20ºC
and a number of samples were also stored at -80ºC. To facilitate haemolysis of
RBCs it is recommended to store a fresh sample for a few hours in a freezer as
freezing destroys the red cells.
2. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge
tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M
NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added.
3. The solution is left for 10 minutes at RT with occasional mixing by inversion
followed by centrifugation for 5 minutes at 4000 rpm.
4. After centrifugation the supernatant is discarded and a white pellet will be
observed at the bottom of the tube. This pellet must be washed for at least 3
times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It
is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer
in order to clean the white blood cells from remaining heme.
5. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing
100µ g/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are
added to the pellet. The tubes are then incubated at a temperature of 37 - 55ºC
(optimal temp for Proteinase K activity) overnight in a water bath or incubator.
During this step the white blood cells' membranes are denatured and DNA goes
out in solution.
6. After the incubation, 1.5mls of SE buffer together with 750µ l of 6M (saturated)
NaCl are added to each tube, followed by the addition of 3.75mls chloroform.
7. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional
mixing for at least 30min. Alternatively you can leave the tubes on a rotator for
1 hour.
8. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal
breaking force. After centrifugation 2 phases are observed and care must be
taken not to disturb the interphase. During this step DNA is extracted into the
supernatant and proteins separated into the lower phase.
9. The upper aqueous phase (containing the DNA) is transferred into a clean and
sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the
addition of an equal volume of isopropanol.
10. DNA will be precipitated by gentle swirling of the tube and is observed visually
as a white thread like strand.
11. Using a sterile spatula or loop transfer the DNA strand into a sterile
microcentrifuge tube containing 1ml of 75% ethanol.
12. The DNA is then washed by inversion to clean it from any remaining salts and
the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded
taking care not to discard the pellet. Repeat this step once more.
13. After discarding the supernatant the pellet is dried from excess ethanol either by
using a vacuum centrifuge or by leaving the tubes open and inverted in an oven
at around 50 - 65ºC for an hour.
14. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH
8.0) and left overnight on a rotator.
15. DNA concentration is determined either by agarose gel electrophoresis or
spectrophotometry and adjusted to the desired concentration by adding more TE
buffer. It is important to note that before adjusting and reading DNA
concentrations one must obtain a homogeneous sample of DNA which is not
quite easy acquired since DNA is very viscous. To adjust to lower concentration
one must used other quantitation methods such as Picogreen (Molecular Probes)
using spectrofluorometry as spectrophotometry is not accurate and sensitive at
very low concentrations.
DNA Preparation from Blood

Section of Cancer Genomics, Genetics Branch, NCI
National Institutes of Health
Reagents
Ammonium chloride (NH4Cl)
Chloroform
Mallinckrodt, Cat. 4440
Ethanol, absolute
HCl
Isoamyl alcohol
Sigma, Cat. I-3643
Isopropanol (2-Propanol)
Phenol
Potassium carbonate (KHCO3)
Proteinase K
EM Science, Gibbstown, WV, Cat. 24568-2 (100 mg)
Sodium acetate
Sodium chloride
Sodium EDTA (Na2EDTA)
Sodium dodecyl sulfate (SDS 10%)
Digene Diagnostics, Inc, Cat. 3400-1016
Tris EDTA buffer, pH 8.0
Preparation
Lysis Buffer
NH4Cl 8.29 g f.c. [155 mM]
KHCO3 1 g f.c. [10 mM]
Na2EDTA 0.034 g or 200 l EDTA 0.5 M f.c. [0.1mM]
Fill to 1000 ml with distilled water
Adjust to pH 7.4 with 1 M HCl or NaOH for each use
Chloroform/Isoamyl alcohol 24:1
Chloroform 24 ml
Isoamylalcohol 1 ml
SE-Buffer
NaCl 4.39 g f.c. [75 mM]
Na2EDTA 8.41 g or 50 ml EDTA 0.5 M f.c. [25 mM]
Fill to 1000 ml with distilled water
Adjust to pH 8.0 with 1 M NaOH for each use
Sodium acetate
3 M Sodium acetate 246 g/L
Adjust to pH 5.2 with CH3COOH
Proteinase K (10mg/ml)
Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at room
temperature
Aliquot and store at –20°C
Procedure
1. To 10 ml whole blood (EDTA, heparin, citrate) add 30 ml lysis
buffer,
shake gently, incubate for 30 min on ice, and centrifuge at 1200 rpm
for 10
min at 4o C.
2. Remove supernatant (blood waste), add 10 ml lysis buffer,
resuspend the
pellet, and centrifuge for 10 min at 4°C (1200 rpm).
3. Remove supernatant (blood waste), add 5 ml SE-buffer, resuspend
the pellet,
and centrifuge for 10 min at 4°C (1200 rpm).
4. Remove supernatant (blood waste).
(It is possible to store the pellet at -80°C. To do so, add 1 ml SE-buffer
and
resuspend the pellet. Use a cryo-tube and centrifuge at 1200 rpm for
10 min
at 4°C. Remove the supernatant and freeze the pellet.)
Add 5 ml SE-buffer and resuspend the pellet, add 40  l proteinase K
(10
mg/ml) and 250  l 20% SDS, shake gently, and incubate overnight at
37°C in
a water bath.
5. Add 5 ml SE-buffer and 10 ml phenol, shake by hand for 10 min,
and
centrifuge at 3000 rpm for 5 min at 10°C.
6. Transfer the supernatant into a new tube, add 10 ml
phenol/chloroform/isoamyl alcohol (25:24:1), shake by hand for 10
min, and
centrifuge at 3000 rpm for 5 min at 10°C.
7. Again transfer the supernatant into a new tube, add 10 ml
chloroform/isoamylalcohol (24:1), shake by hand for 10 min, and
centrifuge at
3000 rpm for 5 min at 10°C.
8. Transfer the supernatant into a new tube, add 300 μl 3 M sodium
acetate (pH
5.2) and 10 ml isopropanol, shake gently until the DNA precipitated,
use a
glass pipette, make a hook over a bunsen burner, and capture the
DNA.
9. Wash the DNA in 70% ethanol and dissolve the DNA in 0.5-1 ml TE-
buffer
overnight at 4°C on a rotating shaker. (If the DNA is not dissolved
leave it
longer at 4°C on the rotating shaker).
10. Measure the DNA concentration in a spectrophotometer
(Pharmacia,
GeneQuant) and run 200 ng on a 1% agarose gel.
DNA Preparation from Lymphoblastoid Cells

Reagents
Chloroform
Ethanol, absolute
Isoamyl Alcohol
Sigma, Cat. I-3643
Phenol buffer
Gibco BRL, Cat. 15513-039
Phosphate Buffered Saline (PBS), 1X
Proteinase K
Sodium acetate, 3 M, pH 5.2
Sodium dodecyl sulfate (SDS), 10%
TAE buffer
Bio Whittaker, Cat. 16-011V
Tris HCl, pH 8.0
Trypsin
Distilled Water
Preparation
DNA buffer
1M Tris HCl, pH 8.0 100 ml
0.5 M EDTA 100 ml
dH2O water 300 ml
Chloroform/Isoamylacohol 24:1
Chloroform 24 ml
Isoamylacohol 1 ml
Procedure
1. Tap cell culture flask by hand and transfer contents to 15 or 50 ml
centrifuge tube.
Centrifuge cultured cells for 10 min at 1200 rpm. Wash cell pellet
twice with
10 ml 1X PBS, centrifuging between washes.
2. Remove supernatant, wash cells twice with 10 ml DNA buffer and
re-suspend in
10 ml DNA buffer; incubate for 10 min on ice.
3. Centrifuge for 10 min at 1200 rpm and remove supernatant. Add 3
ml DNA buffer;
re-suspend the pellet. Add 100 μl Proteinase K (10 mg/ml) and 400 μl
of 10% SDS.
Shake gently and incubate in 45°C waterbath overnight.
4. Add 3.6 ml of phenol. Shake by hand for 10 min (RT); centrifuge for
10 min at 10°C
at 3000 rpm.
5. Transfer supernatant into a new 50 ml tube. Add 1.8 ml phenol and
1.8 ml
chloroform/isoamylalcohol (24:1), shake by hand for 10 min (RT);
centrifuge for 5
min at 10°C (3000 rpm).
6. Transfer supernatant into a new 50 ml tube. Add 3.6 ml
chloroform/isoamylalcohol
and shake by hand for 10 min (RT); centrifuge for 5 min at 10°C (3000
rpm).
7. Transfer supernatant into a new 50 tube. Add 36 μl (1/10) 3 M
sodium acetate
(pH 5.2), mix gently, and add 3 volumes of 100% ethanol; shake
gently until the
DNA has precipitated.
8. Use a sterile glass pipette to transfer the precipitated DNA into 30
ml of 70%
tube. Place on inverting rack and invert for 2 hr to thoroughly rinse.
Transfer DNA
into a sterile eppendorf tube.
9. Centrifuge for 20 min at 14,000 rpm. Dry pellet in a speed vac for 5
min. Dissolve
the DNA in 300  l sterile water and place on a rotating shaker
overnight at 4°C.
10. Measure the DNA concentration and run 1-5  l (approximately
200 ng)
for gel electrophoresis on agarose gel (1%) in 1X TAE buffer.
DNA From Whole Blood for PCR

(from Higuchi, R. (1989) Amplifications 2: 1-3)
1. Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel
blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediately to
prevent clot formation. Store on ice until processing.
2. Add 200 µl Lysis Buffer to each tube and vortex to suspend evenly.
3. Microfuge 25 seconds at 16000xg to pellet nuclei.
4. Remove and discard supernatant and repeat steps 2-4 two more times, or until no hemoglobin
remains.
5. Resuspend nuclear pellet in 100 µl PBND with 60 µg/ml proteinase K and incubate at 55 C for
60 minutes (or overnight, if convenient).
6. Heat samples to 97 C for 10 minutes to inactivate proteinase K.
7. Add 1-5 µl of DNA solution for a 25 µl PCR reaction.
Reagents:
1) Lysis Buffer
0.32 M Sucrose
10mM Tris-HCl (pH 7.5)
5 mM MgCl2
1% v/v Triton X-100
2) PBND (PCR Buffer with Nonionic Detergents)*
50 mM KCl
10 mM Tris-HCl (pH 8.3)
2.5 mM MgCl2
0.1 mg/ml gelatin
0.45% (v/v) Nonidet P40
0.45% (v/v) Tween 20
Autoclave to sterilize and dissolve gelatin
Store frozen
*Add proteinase K (60 µg/ml) immediately prior to use)
Typical 25 µl PCR reaction:

1-5 µl DNA
2.5 µl 10x Perkin Elmer buffer, 1.5 mM MgCl2 (final)
2 µl 2.5 mM dNTP mixture (2.5 mM each dNTP, 200 µM final)
0.5 µl 20 µM forward primer (0.4 µM final)
0.5 µl 20 µM reverse primer (0.4 µM final)
0.1 µl Taq DNA polymerase (can decrease to 0.05 µl)
dH2O to 25 µl
Extraction of DNA from the whole blood

by silica gel.
Introduction
Here we present the new simple and inexpensive procedure in which the pure DNA is
extracted from the whole blood within 15-20 min. The high yield and quality of the
product are provided by use of the selected glass-based sorbent and optimized
composition of lysing and washing solutions.
The traditional procedure for blood DNA isolation is laborious and time- consuming. It
comprises separation of white cells' fraction from the whole blood, lysis with detergent,
digestion with proteinase, extraction with phenol and precipitation of DNA with
alcohol.
Being applied to human tests such complicated procedure leads to elevated risk for
laboratory personnel to be infected by AIDS, hepatitis, cytomegalovirus as well as the
other viruses potentially present in blood. In this respect an approach seems to be very
attractive to treat the whole blood with such a reagent that simultaneously lyses the
blood and denatures the viral particles. Use of the strong chaotropic agent GuSCN for
lysis of human cells followed by sorbtion of DNA to glass powder was described by
Boom (1) and Bush (2). This approach employs the ability of glass-based sorbents to
bind nucleic acids at high salt concentrations and release at low ones.
The great variety of glass sorbents has been reported last years to be used for DNA
extraction from various sources, containing nucleic acids. For reference, isolation of
DNA fragments from agarose gel (4), plasmid DNA from E.coli (3) and genomic DNA
from eukariotic and prokaryotic cells (1) could be brought up. Nevertheless, the whole
blood represents a certain difficulty for immediate DNA extraction due to presence of
large amount of protein in the sample. Thus, white cells containing DNA count for only
about 2% of human blood cells. This was the reason that DNA extracts appeared to be
contaminated by red material, inhibiting PCR amplification, when we tried to apply
available glass based DNA isolation procedures directly to the whole blood.
However, developing an automated device for blood DNA extraction we preferred the
glass-based biochemical concept, what enabled to perform the whole procedure in one
tube. Optimized parameters of the procedure, improved composition of washing
solutions together with the new choice of the glass sorbent resulted in the new short and
inexpensive protocol which provides DNA of perfect quality for enzymatic reactions
and PCR analysis.
Reagents
• The composition of GUANIDINE SOLUTION was: 6 M GuSCN, 20 mM
EDTA, 10 mM Tris-HCl (pH 6.5), 40 g/l Triton X-100 and 10 g/l DTT.
• BIND MIX was prepared by resuspending 4 g of silica gel (Aldrich, #28,851-9)
in 100 ml of GUANIDINE SOLUTION.
• PROPANOL WASH contained of 25% iso-propanol, 25% ethanol, 100 mM
NaCl and 10 mM Tris-HCl (pH 8.0). All reagents can be stored at room
temperature.
Procedure
1. Mix in 2.2 ml minicentrifuge tube 0.5 ml of whole blood (containing 50 mM
EDTA as anticoagulant) with 1.0 ml of BIND MIX.
2. Incubate at room temperature for 3 min.
3. Collect silica gel by short spin (3 sec, 5000 g).
4. Resuspend the pellet in 1.0 ml of GUANIDINE SOLUTION by vortexing.
5. Pellet the sorbent by short spin (at 5000 g).
6. Repeat washing the silica gel with GUANIDINE SOLUTION.
7. Then, wash the silica gel with PROPANOL WASH (twice) and pure ethanol
(once) in the same way.
8. Thoroughly remove ethanol and dry the silica gel under vacuum with heating
applied.
9. Resuspend the silica gel in 100 ul of TE buffer. Elute DNA at 65oC for 3 min.
10. Resuspend the mix again, centrifuge for 10 sec, take the supernatant
Time, yield and quality

The total time of the procedure is 15-20 minutes. The yield of DNA in the procedure is
routinely 40 ug per milliliter of blood. The size of DNA is more than 50 kb. The quality
of DNA provides reliable PCR amplification and enzymatic restriction. Samples can be
stored at room temperature for months with no degradation. Absence of nucleases was
proved by overnight incubation of the DNA at 37oC in presence of 10 mM MgCl2.
Parameters of the procedure

The following new elements have been introduced in the procedure in order to
provide maximal yield and quality of DNA and get rid of contaminants in the
product:
1. The silica gel Aldrich (#28,851-9) was selected as a result of comparative testing
of commercially available glass-based sorbents.
2. Chaotropic salt guanidine thiocyanate was used in combination with nonionic
detergent Triton X-100 at 4% concentration and anti-oxidizing agent DTT at 1%
concentration in GUANIDINE SOLUTION.
3. Iso-propanol and NaCl were included in the composition of alcohol washing
solution.
4. The parameters of the procedure were systematically optimized to provide said
requirements.
Temperature
The procedure is flexible in relation to temperature and time of incubation. If necessary,
the isolation process can be suspended at any step and the tubes can be kept for hours at
room temperature.
If the blood probes are coagulated, the red clots can be preliminary dissolved in
GUANIDINE SOLUTION at 65oC. However sorbtion and washing of DNA can not be
performed at elevated temperature inasmuch as heating of blood-guanidine- silica gel
mix leads to partial cleavage of DNA. At the same time heating doesn't notably affect
binding of DNA to silica gel.
Washing and drying

No DNA can be lost while washing under specified conditions. PROPANOL WASH
removes guanidine and the rest of red material from the silica gel, leaving the purified
DNA bound to the silica gel. Subsequent washing with pure ethanol or acetone as well
as complete removing of liquid is necessary to enable quick drying.
The special care should be taken of preventing cross-contamination of the samples

during drying under vacuum. Drying the tubes on 65oC uncovered solid-state heater is
also available.
Elution
Elution step can be performed at 65oC for 3 min or at room temperature for 15 min.
Then, the samples can be stored at room temperature in the same tubes over the silica
gel.
Thus, the whole procedure can be fulfilled in short time in one tube in manual mode or
in simple disposable plastic cassette in automated or semi-automated mode.
The presented procedure seems to be the most appropriate for use in clinics for multiple
PCR-based diagnostic tests due to its rapidity, simplicity and safety.
Extraction of genomic DNA from whole blood
Reagents
Buffer A (Red blood cell lysis buffer) composition
• 0.32 M sucrose
• 10 mM Tris HCl
• 5 mM MgCl2
• 0.75% Triton-X-100
Adjust pH to 7.6
Buffer B (Proteinase K buffer) composition
• 20 mM Tris-HCl
• 4 mM Na2EDTA
• 100 mM NaCl
Adjust pH to 7.4
N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition
of Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better
option.
Procedure
1. Add 1 volume of buffer A to 1 volume of blood and 2 volumes of cold,

sterile, distilled, deionised water. Vortex gently or invert tube 6-8 times and
leave to incubate on ice for 2-3 minutes.
2. Spin at 3500 rpm for 15 minutes at 4oC. Discard supernatant into 2.5%
bleach solution and re-suspend pellet (vortex for 30 seconds at medium speed)
in 2 ml of buffer A and 6 ml of water. Spin at 3500 rpm for 15 minutes at 4oC.
The pellet should be white to cream in colour. If pellet is significantly red,
repeat washing step again.
3. Add 5 ml of Buffer B and 500 µl of 10% SDS to pellet. Re-suspend pellet by
vortexing vigorously for 30-60 seconds. Then add 50 µl of Proteinase K solution
(20mg/ml). The Proteinase K solution should be made fresh and refrigerated
prior to use.
4. Leave to incubate for two hours at 55oC in a water bath. Remove samples
and leave to cool to room temperature (or leave for 2-3 minutes on ice). Add 4
ml of 5.3 M NaCl solution. Vortex gently for 15 seconds.
5. Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh
tube. Take care not to dislodge pellet. Add an equal volume of cold isopropanol
(stored at -20oC). Invert 5-6 times gently to precipitate DNA.
6. Remove DNA with a wide bore tip and transfer to a microfuge tube. Wash
with 1 ml of 70% ethanol. Leave DNA to dry for 15-20 minutes at 37oC. Re-
suspend in 300-400 µl of Tris HCl, pH 8.5 (not TE!). Leave to re-dissolve
overnight at room temperature. DNA can be safely refrigerated for up to a year.
Long-term storage may involve ethanol at -70oC.
DNA Preparation from Adherent Cells
Reagents
Chloroform
Mallinckrodt, cat. 4440
EDTA, 0.5 M
Ethanol, 100%
Ethanol, 70%
Isoamyl Alcohol
Sigma, cat. I-3643
Phenol
Phosphate Buffered Saline (PBS), 10X and 1X
Proteinase K
Sodium acetate, 3 M, pH 5.2
Sodium dodecyl sulfate (SDS), 10%
Tris-HCl, 1 M, pH 8.0
TAE buffer (Tris acetate/disodium EDTA), 1X
Bio Whittaker, Cat. 16-011V
Trypsin
Distilled Water
Preparation
DNA buffer
1M Tris-HCl, 1 M, pH 8.0 100 ml
0.5 M EDTA 100 ml
dH2O water 300 ml
Chloroform 24 ml
Isoamyl alcohol 1 ml
Procedure
1. Use trypsin or cell scraper to remove cells from tissue culture flask
(T-75). Centrifuge
cultured cells for 10 min at 10°C (1200 rpm). Remove supernatant and
re-suspend
cell pellet in 1X PBS and wash twice with 10 ml 1X PBS, centrifuging
between
washes.
2. Resuspend pellet in 10 ml DNA buffer. Centrifuge cells for 10 min at
10 °C (1200
rpm). Remove supernatant.
3. Add 3 ml DNA-buffer, re-suspend the pellet, add 125 ml Proteinase
K (10 mg/ml) and
400 ml 10% SDS; shake gently and incubate overnight at 45°C.
4. Add 3.6 ml of phenol, shake by hand for 10 minutes (RT);
centrifuge for 10 min at
10°C (3000 rpm).
5. Transfer the supernatant into a new tube (15 ml); measure the
volume. Add 1.8 ml
phenol and 1.8 ml chloroform/isoamylalcohol (24:1) or a total amount
equal to the
volume of the supernatant. Shake by hand for 10 min (RT); centrifuge
for 10 min at
10°C (3000 rpm).
6. Transfer the supernatant into a new tube (15 ml); measure the
volume. Add 3.6 ml
chloroform/isoamylalcohol (24:1) or an amount equal to the volume of
the
supernatant. Shake by hand for10 min (RT); centrifuge for 10 min at
10°C (3000
rpm).
7. Transfer the supernatant into new tube, measure the volume. Add
1/10 volume 3 M
sodium acetate (pH 5.2) and 3 x the volume 100% isopropanol (2-
propanol);
shake gently until the DNA is precipitated.
8. Use a sterile glass pipette to transfer the precipitated DNA into a
tube with 30 ml of
70% ethanol tube. Place on inverting rack and invert for 2 hr to
thoroughly rinse.
Transfer DNA into a sterile eppendorf tube.
9. Centrifuge for 20 min at 14,000 rpm. Dry pellet in a SpeedVac for 5
min. Dissolve
the DNA in 300-500  l sterile water and place in an eppendorf
thermomixer
shaker overnight at 37°C.
10. Measure the DNA concentration and run 1-5  l (approximately
200 ng) for gel
electrophoresis on agarose gel (1%) in 1X TAE buffer.
DNA Preparation from Fresh/Frozen Tissue

Reagents
Chloroform
EDTA, 0.5 M
Ethanol, absolute
Isoamyl alcohol
Sigma, Cat. I-3643
Phenol
Proteinase K
EM Science, Gibbstown, WV Cat. 24568-2 (100 mg)
RNase A
Boehringer Mannheim, Cat. 109 169
Sodium dodecyl sulfate (SDS) solution, 10%
Digene Diagnostics, Beltsville, MD, Cat. 3400-1016
Preparation
DNA buffer (Tris-EDTA)
1 M Tris pH 8.0 20 ml
0.5 M EDTA 20 ml
Sterile water 100 ml
Proteinase K (10mg/ml)
Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at room
temperature
(RT)
RNase A (20 mg/ml)
Dissolve 200 mg RNase A in 10 ml sterile water, boil for 15 min, and
cool to RT.
Procedure
1. Put 60-80 mg of tissue in a petri dish with culture media and divide
the
tissue into two pieces.
2. Put the tissue into two sterile 15 ml tubes and centrifuge for 2 min
at 4°C at
1500 rpm.
3. Remove the supernatant, and wash twice with 1 ml 1X PBS or DNA-
buffer.
(It is possible to store the pellet at -80°C; in that case, add 1 ml 1X
PBS and
resuspend the pellet. Use a cryo-tube and centrifuge at 1500 rpm for
2 min at
4°C. Remove the supernatant, and freeze the pellet.)
4. Remove supernatant and resuspend the pellet in 2.06 ml DNA-
buffer.
5. Add 100 μl proteinase K (10 mg/ml) and 240 μl 10% SDS, shake
gently, and
incubate overnight at 45°C in a waterbath.
6. If there are still some tissue pieces visible, add proteinase K again,
shake
gently, and incubate for another 5 hr at 45°C.
7. Add 2.4 ml of phenol, shake by hand for 5-10 min, and centrifuge
at 3000 rpm
for 5 min at 10°C.
8. Pipette the supernatant into a new tube, add 1.2 ml phenol, and
1.2 ml
chloroform/isoamyl alcohol (24:1); shake by hand for 5-10 min, and
centrifuge
at 3000 rpm for 5 min at 10°C.
9. Pipette the supernatant into a new tube, add 2.4 ml
chloroform/isoamyl
alcohol (24:1), shake by hand for 5-10 min, and centrifuge at 3000
rpm for 5
min at 10°C.
10. Pipette the supernatant into a new tube, add 25 μl 3 M sodium
acetate (pH
5.2) and 5 ml ethanol, shake gently until the DNA precipitates.
11. Take a glass pipette, heat it over a gas burner, and bend the end
to a hook. Fish
the DNA thread out of the solution using the hook and transfer DNA to
a new
tube.
12. Wash the DNA in 70% ethanol and dry it in the speed vac.
13. Dissolve the DNA in 0.5-1 ml sterile water overnight (or longer if
necessary)
at 4°C on a rotating shaker.
14. Measure the DNA concentration in a spectrophotometer and run
200 ng on a 1% agarose gel.
Tissue (mg) 5 10 15 20 40 60 80 100
Volume in μl
Total 400 800 1200 1800 3200 4800 6400 8000
DNA buffer 360 680 1020 1360 2720 4080 5440 6800
Proteinase 20 40 60 80 160 240 320 400
10% SDS 40 80 120 160 320 480 640 800
DNA ISOLATION FROM PRIMARY TUMORS VIA CRYOSECTIONS
- make a 5µm section to do an evaluation of the % tumour cells
- make 50X50µm sections for the DNA isolation
- put sections in a falcon tube containing 10 ml of 1XSE
- add 100µg/ml prot. K (endconcentration) (1 ml of 1mg/ml in 10 ml of SE buffer) + 1%

SDS (endconcentration) (stock is 25% SDS)
- put the tube o/n at 55°C
- add 1/4 of the volume (2.5 ml) of 6M NaCl(saturated solution, precipitation at the
bottom)
- forming of precipitate in the tube, mix gently by inverting the tube a few times.
- add 1 vol chloroform (stabilized with ethanol) (12.5 ml) and invert the tube a few
times
- put tube for 1 hr on rotator at room temperature
- centrifuge 10 min 2000 rpm
- forming of 2 phases, phase at the bottom is chloroform, middle phase contains

proteins, SDS,...., top phase contains DNA
- transfer of the top phase to another tube by means of a pipet tip (cut off)
- add 1 vol isopropanol
- invert the tube a few times, thread of DNA will form, if not centrifuge for 30 min 4000
rpm at 4°C
- remove thread of DNA with a closed hooked pasteur pipet and rinse gently in 70%
EtOH
- DNA is dried and dissolved in appropriate volume of TE buffer
- dissolve the DNA o/n at 4°C on rotor
SE buffer (10X) :
750 mM NaCl pH=8
250 mM EDTA
DNA Preparation from Cell Lines, High Salt

Method
Reagents
EDTA
Ethanol, absolute
Isopropanol
Proteinase K
EM Science, Cat. 24568-2
Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)
Quality Biological, Inc., Cat. 351-035-060
Sodium chloride (NaCl)
Sodium Dodecyl Sulfate (SDS ), 10%
Digent Diagnostics, Inc., Cat. 3400-1016
Tris EDTA (TE), pH 7.4
Tris EDTA (TE), pH 8.0
Tris HCl, pH 8.0
Quality Biological, Inc, Cat. 351-007-100
Preparation
Proteinase K solution
Proteinase K 100 mg
Tris EDTA, pH 7.4 10 ml
Nuclei lysis buffer
Tris EDTA, pH 8.0 1 ml
NaCl, 5 M 8 ml
EDTA, 0.5 M 0.4 ml
Add dH2O to 100 ml
6 M Sodium chloride
Sodium chloride 3.5 g
dH2O 10 ml
Procedure
A. Cell Collection and Lysis
1. Use trypsin or cell scraper to remove cells from tissue culture flask
(T-75).
Centrifuge cultured cells for 10 min at 10°C (1200 rpm). Remove and
resuspend cell pellet twice with 10 ml 1X PBS, centrifuging between
washes.
2. Resuspend pellet in 10 ml nuclei lysis buffer. Centrifuge cultured
cells
for 10 min at 10°C (1200 rpm). Remove supernatant.
3. Add 3 ml nuclei lysis buffer, resuspend the pellet, add 100  l
Proteinase K
(10 mg/ml) and add 400  l of 10% SDS, shake gently, and incubate
at
45°C overnight.
B. Precipitation in High Salt Concentration
1. To lysate, add 1 ml of 6 M NaCl.
2. Shake tubes vigorously by hand for 15 sec.
3. Centifuge at 3000 rpm for 15 min.
4. Transfer supernatant into a new tube and centrifuge at 3000 rpm
for 15 min.
5. Repeat steps 3 and 4 until tube is clear of salt (at least 3-4 times).
C. Precipitation with Ethanol
1. Transfer supernatant into a new tube; measure the volume of the
supernatant.
2. Add 1/10 the total volume 3 M sodium acetate (pH 5.2) and 2.5-3
times total
volume cold 100% isopropanol; shake gently until the DNA is
precipitated.
3. Using the hook, transfer the DNA into a new tube containing 13 ml
of 70%
ethanol.
4. Place on inverting rack and invert for 2 hr to thoroughly rinse.
5. Transfer DNA into new Eppendorf tube (1.5 ml) and centrifuge for
30 min at
14,000 rpm.
3
6. Dry pellet by inverting on paper, and speed vac for 5 min.
7. Add 200 μl dH20 and resuspend at 37°C overnight in Thermomixer.
8. Measure the DNA concentration and run 1-5  l (approximately 200
ng)
for gel electrophoresis on agarose gel (1%) in 1X TAE buffer. Also,
measure
the DNA with NanoDrop and print out results for future reference.
ES CELL DNA EXTRACTION: TUBE METHOD
Protocol for extracting DNA from ES Cells, starting from the 96-well plate but
processing in an eppendorf tube to recover more of the DNA. NOTE- THIS TAKES A
LOT OF TIME if you do the whole plate this way!
Lysis buffer: ( 100 ml Recipe )

10 mM Tris-HCl pH7.5 - 0.5 ml of 2M
10 mM EDTA - 2 ml of 0.5M
10 mM NaCl - 0.2 ml of 5M
0.5% (w/v) Sarkosyl - 0.5gm (N-Lauroylsarcosine, Sigma # L-9150)
1mg/ml Proteinase K - add fresh each time (stored in freezer).
1. Add 250µl Lysis Buffer (with proteinase K added) to each well.
2. Incubate the plate in a sealed humid container at 60°ree;C for 1 hour. (Meanwhile,
label eppy tubes!)
3. Transfer the contents of each well into a separate 1.5ml eppendorf tube (µ-cent. tube).
4. Continue lysing the cells for 2 more hours. (60°ree;C). (Float tubes in waterbath-
don't need tupperware).
5. Add an equal volume (250 µl) of phenol:chloroform (phenol: chloroform:

isoamylalcohol, 25:24:1).
(The pH of the phenol:chloroform needs to be ~7.8 - 8.0, or else the DNA will partition
into the organic phase).
6. Mix the contents of the tube until an emulsion forms. (Shake by hand for ~1 min, (or
vortex ~2sec),because vortexing shears long (genomic) DNA).
7. Centrifuge 5-10 min in a microfuge at TRm. (The phases should be well-separated).
8. Transfer (pipet) the aqueous phase (the top phase) to a fresh tube. (Toss the
interface/organic phase-- chemical waste in hood).
9. Repeat steps 5-8 until no protein is visible at the interface of the aqueous and organic
phases.
10. Add an equal volume (250 µl) of chloroform and repeat steps 6-8.
11. Optional: Repeat chloroform extraction. (The chloroform extraction removes the
traces of phenol).
12. Add 1/25th volume of 5M NaCl (10µl of 5M NaCl for 250 µl ), for a final
concentration of 0.2M NaCl. Mix well. (use NaCl rather than other salts because of
detergent in lysis buffer).
13. Add exactly 2 volumes of ice-cold ethanol and again mix the solution well.
14. Store the ethanolic solution on ice for 30 min to precipitate the DNA. (can be stored
indefinitely at 0°ree;C or at -20°ree;C).
15. Centrifuge at 12,000xg for 10 min, 0°ree;C. (Try 4°ree;C, microfuge at max
(16,000), 10 min). (Remember to point tube hinges out, to locate pellet even if it's
invisible).
16. Suck off the supernatant (dispo pipet tip, with vacuum flask). Do not disturb the
pellet. Vacuum droplets from walls of tube as well.
17. Half fill the tube with 70% EtOH and re-spin for 2 minutes at 4°ree;C.
18. Suck off supernatant as in step 16.
19. Store the open tube on the bench at TRm until the last traces of fluid have
evaporated. (i.e.: Air dry).
20. Dissolve the DNA in 30µl (or 50 µl, and use half of sample) of 10mM Tris pH 8.5
(or TE). (pipet around the walls of the tube to dissolve the DNA off the walls of the
tube). Let dissolve for at least an hour at 37°ree;C.
21. Restriction digest:

- DNA in Tris pH8.5, from above protocol
- restriction enzyme
- restriction enzyme buffer
- X (BSA if required by enzyme)
- spermidine (to a final conc. of 1mM; stored in freezer)
- (also add: ) DTT to a final conc. of 1mM
Final volume = 40µl or less.
Digest overnight at 37°ree;C.
Mouse and Human Genomic DNA Extraction
Treat human blood and supernatants as potentially infectious. Discard supernatants by

an appropriate method, eg into bromochlor solution.
DNA extraction from frozen blood:

1. Collect 10ml blood into a heparin or EDTA tube.
2. Transfer to a 50ml falcon tube and freeze overnight at -20°C.
3. Allow to thaw in iced water for several hours.
4. Add 40ml of triton/sucrose lysis buffer to blood and invert to mix. Spin samples
at 3000rpm for 15 minutes. Discard supernatant.
5. Add 20ml of triton/sucrose lysis buffer and resuspend white cell pellet with a
blue tip. Spin samples at 3000rpm for 10 minutes. Discard supernatant.
6. Add 3ml salting out/lysis buffer to the white cell pellet. Resuspend using a blue
tip. Add 200µl 20% SDS and 300µl 5mg/ml proteinase K. Incubate samples
overnight at 50°C in a gently shaking incubator. If sample has not been
completely digested overnight, add 50µl 5mg/ml proteinase K and continue
incubation for several more hours.
7. Add 1.5ml 6M NaCl and shake vigorously for 15 seconds. Spin sample at
3000rpm for 15 minutes.
8. Transfer supernatant to a new tube, and add 10ml absolute ethanol. Allow the
DNA to come out of solution. Hook out DNA and transfer to an ependorf tube.
Spin briefly and remove ethanol. Add 400µl TE and allow DNA to resuspend
overnight at 4°C.
Extract once with phenol/chloroform and once with chloroform. Ethanol precipitate and
rinse pellet with 70% ethanol. Allow pellet to resuspend overnight at 4°C.
Triton/sucrose lysis buffer1 litre:
1M MgCl2 5ml
1M Tris-Cl pH 7.5 10ml
Triton X-100 10ml
sucrose 109.54g
Salting out/lysis buffer100mls:
1M Tris-Cl pH 7.5 1ml

5M NaCl 8mls
500mM EDTA pH 8 400µl
Mouse:
1. Dissect one lobe of liver tissue, and freeze in liquid nitrogen.

2. Grind tissue into a fine powder under liquid nitrogen.
3. Transfer tissue to a cold 15 ml blue top tube, add 5 ml of salting out/lysis buffer
(10 mM Tris-Cl pH 7.5, 400 mM NaCl, 2 mM EDTA pH 8) and resuspend
tissue with a blue tip.
4. Add 200 µl 20% SDS and 300 µl 5 mg/ml proteinase K. Incubate samples for
5. several hours at 50°C, until tissue had been completely digested.
Extraction and precipitation:
1. Add 5 ml phenol equilibrated with 500 mM Tris pH 8, place on slowly rotating

wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous
phase to another tube.
2. Add 5 ml phenol/chloroform equilibrated with 500 mM Tris pH 8, place on
slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes.
Remove aqueous phase to another tube.
Add 5 ml chloroform equilibrated with TE, place on slowly rotating wheel for 15
minutes.
3. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
4. Add 0.5 vol isopropanol and allow DNA to come out of solution. Gently swirl
the tube for a few minutes. (**At this stage, tube can be placed at -20°C
overnight.)
5. Hook out the DNA with a pasteur pipette hook, and allow excess solution to
drain from the DNA by holding the blob to the side of the tube.
6. Place hook with DNA on it into a 15 ml blue top tube with 1 ml TE. Break off
pasteur pipette and leave hook with DNA in tube. Place on very gently rocking
platform for several hours until DNA has completely gone into solution.
Quantitate and resuspend DNA at a final concentration of 500 µg./ml.
A. Phenol extraction of DNA samples

Phenol extraction is a common technique used to purify a DNA sample (1). Typically,
an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a
microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact
phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding
the phenol interface and then is subjected to two ether extractions to remove residual
phenol. An equal volume of water-saturated ether is added to the tube, the mixture is
vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is
removed and discarded, including phenol droplets at the interface. After this extraction
is repeated, the DNA is concentrated by ethanol precipitation.
Protocol
1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5
ml microcentrifuge tube and vortex for 15-30 seconds.
2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.
3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding
proteins at the aqueous:phenol interface. At this stage the aqueous phase can be
extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform,
centrifuged and removed to a clean tube as above but this additional extraction usually
is not necessary if care is taken during the first phenol extraction.
4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3
minutes at room temperature. Remove and discard the upper, ether layer, taking care to
remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.
5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as

discussed below.
B. Concentration of DNA by ethanol precipitation

Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in
a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes.
Frequently, this precipitation is performed by incubation at -20C overnight (1). To
recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the
DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation,
the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.
Protocol
1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample
contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water
bath for at least 10 minutes. It is possible to place the sample at -20degC overnight at
this stage.
2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes at 4 degC,

decant the supernatant, and drain inverted on a paper towel.
3. Add 80% ethanol (corresponding to about two volume of the original sample),
incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and
decant and drain the tube, as above.
4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes,
or until dry.
5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (termed

10:0.1 TE buffer).
6. It is advisable to aliquot the DNA purified in large scale isolations (i.e. 100 ug or
more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because
repeated freezing and thawing is not advisable.
Notes on precipitation of nucleic acids
A. General rules
Most nucleic acids may be precipitated by addition of monovalent cations and two to
three volumes of cold 95% ethanol, followed by incubation at 0 to -70 degC. The DNA
or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at
4degC. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes
residual salt and moisture.
The general procedure for precipitating DNA and RNA is:
1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be

precipitated,
2. Add two volumes of cold 95% ethanol,
3. Place at -70degC for at least 30 minutes, or at -20degC overnight.
or alternatively
1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile
water. Mix by inversion and store at -20degC.
2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be
precipitated.
3. Place at at -70degC for at least 30 minutes or -20degC for two hours to overnight.
* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA
also may be precipitated by addition of 0.6 volumes of isopropanol.
B. Oligonucleotides
Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol.
Place at -70degC for at least one hour.
C. RNA
Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol.
Precipitate large volumes at -20degC overnight.
Small volume samples may be precipitated by placing in powdered dry ice or dry ice-
ethanol bath for five to 10 minutes.
D. Isobutanol concentration of DNA
DNA samples may be concentrated by extraction with isobutanol. Add slightly more
than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases.
Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl
ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated
as described above.
E. Notes on phenol extraction of nucleic acids
The standard and preferred way to remove proteins from nucleic acid solutions is by
extraction with neutralized phenol or phenol/chloroform. Generally, samples are
extracted by addition of one-half volume of neutralized (with TE buffer, pH 7.5) phenol
to the sample, followed by vigorous mixing for a few seconds to form an emulsion.
Following centrifugation for a few minutes, the aqueous (top) phase containing the
nucleic acid is recovered and transferred to a clean tube. Residual phenol then is
removed by extraction with an equal volume of water-saturated diethyl ether. Following
centrifugation to separate the phases, the ether (upper) phase is discarded and the
nucleic acid is ethanol precipitated as described above.
A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from
nucleic acid samples. Following extraction with phenol/chloroform, the sample should
be extracted once with an equal volume of chloroform, and ethanol precipitated as
described above.
C. Restriction digestion
Restriction enzyme digestions are performed by incubating double-stranded DNA
molecules with an appropriate amount of restriction enzyme, in its respective buffer as
recommended by the supplier, and at the optimal temperature for that specific enzyme.
The optimal sodium chloride concentration in the reaction varies for different enzymes,
and a set of three standard buffers containing three concentrations of sodium chloride
are prepared and used when necessary. Typical digestions included a unit of enzyme per
microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is
defined as the amount of enzyme needed to completely digest one microgram of double-
stranded DNA in one hour at the appropriate temperature. These reactions usually are
incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for
enzyme activity, typically 37degC. See the Appendix for a listing of restriction sites
present in the M13 (pUC) MCS and a listing of various restriction enzymes, incubation
conditions and cut sites.
Protocol
1. Prepare the reaction for restriction digestion by adding the following reagents in the
order listed to a microcentrifuge tube:
sterile ddH20 q.s (where "q.s." means quantity sufficient)

10X assay buffer one-tenth volume
DNA x ul
restriction enzyme* y ul (1-10 units per ug DNA)
Total volume z ul
*If desired, more than one enzyme can be included in the digest if both enzymes are
active in the same buffer and the same incubation temperature.
Note: The volume of the reaction depends on the amount and size of the DNA being
digested. Larger DNAs should be digested in larger total volumes (between 50-100 ul),
as should greater amounts of DNA.
Refer to the vendor's catalog for the chart of enzyme activity in a range of salt
concentrations to choose the appropriate assay buffer (10X High, 10X Medium, or 10X
Low Salt Buffers, or 10X SmaI Buffer for SmaI digestions). Restriction enzymes are
purchased from Bethesda Research Laboratories, New England Biolabs, or United
States Biochemicals.
2. Gently mix by pipetting and incubate the reaction at the appropriate temperature
(typically 37degC) for 1-3 hours.
3. Inactivate the enzyme(s) by heating at 70-100degC for 10 minutes or by phenol

extraction (see the vendor's catalog to determine the degree of heat inactivation for a
given enzyme). Prior to use in further protocols such as dephosphorylation or ligation,
an aliquot of the digestion should be assayed by agarose gel electrophoresis versus non-
digested DNA and a size marker, if necessary.
D. Agarose gel electrophoresis

Agarose gel electrophoresis (2) is employed to check the progression of a restriction
enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR
reaction, and to size fractionate DNA molecules, which then could be eluted from the
gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm
gel solution then is poured into a mold (made by wrapping clear tape around and
extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-
forming comb. The percentage of agarose in the gel varied. Although 0.7% agarose gels
typically are used, in cases where the accurate size fractionation of DNA molecules
smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the
expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to
enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are
submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA
samples are mixed with gel tracking dye and loaded into the sample wells.
Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature,
depending on the desired separation. When low-melting agarose is used for preparative
agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the
desired separation, and a fan is positioned such that the heat generated is rapidly
dissipated. Size markers are co-electrophoresed with DNA samples, when appropriate
for fragment size determination. Two size markers are used, phi-X 174 cleaved with
restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda
phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23
kb. After electrophoresis, the gel is placed on a UV light box and a picture of the
fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid
camera.
Protocol
1. Prepare an agarose gel, according to recipes listed below, by combining the agarose
(low gel temperature agarose may also be used) and water in a 500 ml Ehrlenmeyer
flask, and heating in a microwave for 2-4 minutes until the agarose is dissolved.
0.7% 1.0% 2.0%

agarose 1.05 g 1.5 g 3.0 g
20X TAE 7.5 ml 7.5 ml 7.5 ml
ddH2O 142.5 ml 142.5 ml 142.5 ml
EtBr (5 mg/ml) 25 ul 25 ul 25 ul
total vol 150 ml 150 ml 150 ml

Genetic technology grade (800669) or low gel temperature (800259) agarose from
Schwarz/Mann Biotech.
2. Add 20X TAE and ethidium bromide (EtBr), swirl to mix, and pour the gel onto a
taped plate with casting combs in place. Allow 20-30 minutes for solidification.
3. Carefully remove the tape and the gel casting combs and place the gel in a horizontal
electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the
buffer just covers the agarose gel.
4. Add at least one-tenth volume of 10X agarose gel loading dye to each DNA sample,
mix, and load into the wells. Electrophorese the gel at 150-200 mA until the required
separation has been achieved, usually 0.5-1 hour (100-120 mA for low gel temperature
agarose), and cool the gel during electrophoresis with a fan. Visualize the DNA
fragments on a long wave UV light box and photograph with a Polaroid camera.
E. Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an
unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised
with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then
melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture
again is frozen and then thawed. After this second thawing, the tube is centrifuged and
the aqueous layer removed to a new tube. Residual phenol is removed with two ether
extractions, and the DNA is concentrated by ethanol precipitation.
Protocol
1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and
freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this
stage in the elution procedure and leave the gel slice frozen at -70degC.
2. Melt the slice by incubating the tube at 65degC.
3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the
sample at -70degC for 15 minutes.
4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at
room temperature to separate the phases. The aqueous phase then is removed to a clean
tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet
is rinsed and dried.
F. Kinase end-labeling of DNA

Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-
rATP, T4 polynucleotide kinase, and buffer (3). After incubation at 37degC, reactions
are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with
gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The
gel percentage and electrophoresis conditions varied depending on the sizes of the DNA
molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film,
as discussed below for radiolabeled DNA sequencing.
Protocol
1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:
sterile ddH2O q.s

10X kinase buffer 1 ul
DNA x ul
[[gamma]]-[32-P]-rATP 10 uCi
T4 polynucleotide kinase 1 ul (3U/ul)
10 ul
[[gamma]]-[32-P]-rATP (35020) ICN and T4 polynucleotide kinase (70031) from

United States Biochemicals.
2. Incubate at 37degC for 30-60 minutes.
3. Heat the reaction at 65degC for 10 minutes to inactivate the kinase.
G. Bacterial cell maintenance

Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation
(4), XL1BMRF' (Stratagene) for M13 or pUC-based DNA transformation (5), and
ED8767 for cosmid DNA transformation (6-8). To maintain their respective F' episomes
necessary for M13 viral infection (9), JM101 is streaked onto a M9 minimal media
(modified from that given in reference (1) plate and XL1BMRF' is streaked onto an LB
plate (1) containing tetracycline. ED8767 is streaked onto an LB plate. These plates are
incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid media are
inoculated with a smear of several colonies and incubated at 37degC for 8 hours, and
those cultures then are transferred into 50 ml of respective liquid media and further
incubated 12-16 hours. Glycerol is added to a final concentration of 20%, and the
glycerol stock cultures are distributed in 1.3 ml aliquots and frozen at -70degC until use
(1).
Protocol
1. Streak a culture of the bacterial cell strain onto an agar plate of the respective
medium, listed below, and incubate at 37degC overnight.
E. coli strain Agar Medium/Liquid Media

XL1BMRF' (Stratagene) LB-Tet
JM101 M9
ED8767 LB
2. Pick several colonies into a 12 X 75 mm Falcon tube containing a 2 ml aliquot of the
respective liquid media, and incubate for 8-10 hours at 37degC with shaking at 250
rpm.
3. Transfer the 2 ml culture into an Ehrlenmeyer flask containing 50 ml of the

respective liquid media and further incubate overnight (12-16 hours) at 37degC with
shaking at 250 rpm.
4. Add 12.5 ml of sterile glycerol for a final concentration of 20%, and distribute the
culture in 1.3 ml aliquots into 12 X 75 mm Falcon tubes.
5. Store glycerol cell stocks frozen at -70degC until use.
Notes on Restriction/Modification Bacterial Strains:
1. EcoK (alternate=EcoB)-hsdRMS genes=attack DNA not protected by adenine

methylation. (ED8767 is EcoK methylation -). (10)
2. mcrA (modified cytosine restriction), mcrBC, and mrr=methylation requiring systems

that attack DNA only when it IS methylated (Ed8767 is mrr+, so methylated adenines
will be restricted. Clone can carry methylation activity.) (10)
3. In general, it is best to use a strain lacking Mcr and Mrr systems when cloning
genomic DNA from an organism with methylcytosine such as mammals, higher plants ,
and many prokaryotes.(11)
4. The use of D(mrr-hsd-mcrB) hosts=general methylation tolerance and suitability for

clones with N6 methyladenine as well as 5mC (as with bacterial DNAs). (12)
5. XL1-Blue MRF'=D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1,

recA, gyrA96, relA1, lac, l-, [F' proAB, lacIqZDM15, Tn10(tetr)].
Rapid Isolation of Genomic DNA from Human and

bovine Oral Mucosa
Procedure
1. Take an oral smear

2. Dissolve in 50 ml H2O through vigorous shaking
3. Pellet cells 5 min at 4000 rpm
4. Re-suspended the pellet in 400 μl Lysis buffer (50 mM TRIS-Cl pH 8, 10 mM EDTA, 2%
SDS)
5. Incubate for 5 min at 65¡ãC
6. Add 250 μl 4.5 M NaCl and mix
7. Pellet cellular debris (13000 rpm for 4 min). Genomic DNA stays in the supernatant
8. Precipitate DNA by adding 650 μl of isopropanol to the supernatant and centrifuge 5 min at
13000 rpm
9. Re-suspend the DNA in an appropriate buffer (e.g. TrisCl, pH=8.0)
Salting Out Procedure for Human DNA Extraction
April 24, 1990

C. Helms
Principle:
This procedure avoids using phenol and chloroform by using high salt
concentrations to remove proteins. It is rapid, safe and inexpensive. The
procedure is written for the quantity of white blood cells obtained from one or
two tubes of blood, and should be considered a miniprep for human DNA.
Average yields are similar to that obtained with the phenol-chloroform
extraction procedure (50- 200 ug), and the quality of DNA is excellent. The
procedure may be scaled up to handle larger samples.
Time required:
2 days
Special solutions:
• Saturated NaCl solution
Procedure:
Day 1
1. Isolate nuclei from 1-2 tubes of blood (collected in ACD or EDTA tubes):
Add 9 volumes Buffer A, mix well and hold on ice 2 minutes. Spin at 1500 rpm
at 4deg.C for 15 minutes. Resuspend the nuclei pellet in 5 ml Buffer B and
transfer to a 15 ml polypropylene centrifuge tube.
2. Add 500 ul 10% SDS and 55 ul proteinase K (10 mg/ml stock). Incubate at
37deg.C overnight on a low-speed rocker or orbital shaker.
Day 2
1. Add 1.4 ml saturated NaCl soution (approximately 6M) to each tube. Shake
vigorously for 15 seconds. Spin the tubes at 2500 rpm in the Beckman low speed
centrifuge for 15 minutes.
2. Transfer the supernatant to another 15 ml polypropylene tube, leaving behind
the precipitated protein pellet.
3. Add exactly two volumes of room temperature 100% ethanol and invert the tube
several times until the DNA precipitate is visible.
4. Remove the DNA strands with a plastic spatula or pipet tip and transfer to an
eppendorf tube containing 100 - 200 ul TE. Allow the DNA to dissolve at least 2
hours at 37deg.C before quantitating.
Solutions:
• Buffer A
0.32 M sucrose 109.5 g sucrose 10 mM Tris HCl pH
7.6 10 ml 1 M Tris-HCl pH 7.6 5 mM MgCl2 5
ml 1M MgCl2 1 % Triton-X-100 bring volume to 1 liter with
deionized water Sterilize above solution by autoclaving, then
add 10 ml Triton-X-100.
• Buffer B
25 mM EDTA pH 8.0 50 ml EDTA pH 8.0 75 mM NaCl
40 ml 5 M NaCl Bring volume to 1 liter with deionized water.
Sterilize by autoclaving.
• Saturated NaCl solution ( approximately 6M)
Dissolve 35 grams of NaCl in a total volume of 100 ml (deionized water). If the

solution has no precipitate, add 2 grams NaCl and mix. Repeat until no more
NaCl will go into solution. Filter sterilize.
Simplified DNA Extraction from Cell or Tissue

Purpose
DNA extraction without phenol extraction and centrifugation.
Procedure
1. Lysis: the lysis buffer (usually 0.5ml) is added to the tissue or cell ( for a 75cm2
flask, add 5ml directly to the cell). Digestion is complete within several hours at
37C (cell, 2-3h) or 55C (tissue) with agitation.
Lysis buffer
100mM Tris Hcl pH 8.5 5ml
0.5M EDTA 0.5ml
10% SDS 1ml
5M NaCl 2ml
20mg/ml Proteinase K 0.25ml
Bring up to 50ml with dd water
2. isopropanol precipitation: one volume of isopropanol is added to the lysate and

the samples are mixed or swirled until precipitation is complete ( about 10-20 min )
(viscosity completely gone).
3. Recovery of precipitate: the DNA is recovered by lifting the aggregated

precipitate from the solution using a disposable yellow tip. Excess liquid is dabbed
off and the DNA is dispersed in a prelabeled Eppendorf tube containing, depending
on the size of the precipitate, 20 to 500ul to 10mM Tris HCl, 0.1mM EDTA, pH 7.5.
complete dissolution of the DNA may requires several hours of agitation at 37C or
55C (may need overnight). It is important that the DNA is completely dissolved to
ensure the reproducible removal of aliquots for analysis.
DNA Preparation from Paraffin Tissue
Reagents
Chloroform
EDTA, 0.5 M
Ethanol, absolute
Isoamyl alcohol
Sigma, Cat. I-3643
Phenol
Proteinase K
RNase A
Boehringer, Cat. 109 169
Sodium acetate, pH 5.2
Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M
Sigma, Cat. S 7757, 250 g)
TE Buffer (Tris-EDTA), pH 7.4
Tween 20
Xylene
Preparation
Chloroform 24 ml
Isoamyl alcohol 1 ml
DNA extraction buffer
1.5 ml 5M NaCL
5.0 ml 0.5M EDTA
0.5 ml Tween 20
Fill up to 100 ml with sterile water.
Proteinase K (10 mg/ml)
Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at RT.
Aliquot and store at –20°.
2
RNase A (20 mg/ml)
Dissolve 200 mg RNase A in 5 ml sterile water.
Boil for 15 min.
Cool to room temperature
Aliquot and store at –20°.
Procedure
1. Cut 50  m slices of formalin-fixed and paraffin-embedded tumor
samples.
(Note: Cut 4  m slices before and after each 50  m slice for
Haematoxylin-
Eosin staining to insure that the tissue is still representative.)
2. Incubate in xylene at 45°C for 15 min.
3. Centrifuge 10 min at 14,000 rpm.
4. Pipet off supernatant.
5. Repeat steps 2-4.
6. Add 1 ml 100% ethanol to tissue pellet, vortex, centrifuge for 10
min at
14,000 rpm, and pipet off supernatant.
7. Add 1 ml 90% ethanol to tissue pellet, vortex, centrifuge for 10 min
at
14,000 rpm, and pipet off supernatant.
8. Add 1 ml 70% ethanol to tissue pellet, vortex, centrifuge for 10 min
at 14,000
rpm, pipet off supernatant, and dry pellet in speed vac.
9. Resuspend pellet in l ml NaSCN (1 M) and incubate at 37°C
overnight.
10. Centrifuge for 10 min at 14,000 rpm; pipet off supernatant.
11. Resuspend pellet in 400  l of DNA extraction buffer.
12. Add 5 μl RNase (20 mg/ml) and incubate for 1 hr at 37°C (RNase
treatment is
optional, paraffin material often does not contain large amounts of
RNA).
13. Add 40 μl of Proteinase K (10 mg/ml), vortex briefly, and incubate
at 55°C
overnight (if tissue is not completely dissolved, add additional
proteinase K
and continue incubating; tissue should be dissolved).
3
14. Add 440 μl of phenol, shake vigorously by hand for 5 min, and
centrifuge for
5 min at 8000 rpm.
15. Pipet supernatant into a new tube, add a solution of 220 μl phenol
plus 220 μl
chloroform/isoamyl alcohol (24:1), shake vigorously by hand for 5
min, and
centrifuge for 5 min at 8000 rpm.
16. Pipet supernatant into a new tube, add 440 μl chloroform/isoamyl
alcohol
(24:1), shake vigorously by hand for 5 min, and centrifuge for 5 min
at 8000
rpm.
17. Pipet supernatant into a new tube (2 ml eppendorf tube), add
1/10 volume of
sodium acetate (pH 5.2), add 3 volumes of ice cold 100 % ethanol,
and keep
tube for 1 hr at –80°C or overnight at –20°C.
18. Centrifuge for 30 min at 4°C.
19. Remove and save supernatant (optional, in case you are not sure
of the
precipitation).
20. Dry pellet in speed vac.
21. Add 20-50 μl sterile H20 (depending on the amount of DNA you
expect,
which is subjective to experience).
22. Shake gently in thermomixer at 37°C for 2 hours (DNA should be
dissolved,
but if you have doubts put on a rotating shaker in the cold room
overnight).
23. Measure DNA concentration with a spectrophotometer and run
around 200 ng
on a 1% agarose gel.
PROTOCOL TO EXTRACT DNA FROM PARAFFIN BLOCKS
1. Cut 10-20X 10 um sections of formalin fixed paraffin samples into eppendorf

tubes.
2. Add 1 ml xylene, mix, incubate at 55 C for 15 mins. Release pressure, spin
down for 2 minutes in eppendorf ultramicrofuge, pipet off & discard
supernatent.
3. Repeat Step 2.
4. Add 100% ethanol, incubate for 15 mins. Spin down.
5. Remove ethanol, Repeat Step 4, allow pellet to air dry.
6. Add up to 1 ml of proteinase K in digestion buffer to a final concentration of
0.3-0.5 mg/ml (less buffer for smaller tumors).
7. Incubate overnight, shaking, at 55 C.
8. Add fresh concentrated PK (same amount as day 1 from 20 mg/ml stock).
9. Incubate overnight at 55 C.
10. Repeat step 8 & 9.
11. Add equal volume PCI to the PK digested aqueous solution, spin 2 mins, remove
upper aqueoues phase to new tube. (repeat PCI extraction if necessary -- it
usually is).
12. Take 330 ul aqueous phase per eppendorf tube, add 1/2 original volume (165ul)
ammonium acetate (7.5M) [can add 1-3 ul Glycogen here to preserve low
amounts of DNA. Glycogen makes a nice visible pellet] and 2-2.5X volume
with 100% ethanol. Let sit at RT for ~2 hours or overnight at -20 C.(better
O.N.).
13. Spin for 20 minutes at 4 C, decant ethanol (rinse pellet with etoh if it doesn't
move), blot dry, air dry.
14. Carefully resuspend the pellets in small volumes of TE buffer (~15-20 ul) and
let dissolve at RT overnight (or 55 C for 2 hours). Combine tubes from the same
sample.
15. Measure DNA concentration on the Fluorometer. Ideal DNA concentration is
from 200-500 ug/ml.
16. Denature the DNA at 70 C before running 0.2 ug on 1% gel to check the size.
Size should range from 100bp-3Kb.
Solutions
Digestion buffer: 100 mM NaCl/10mM Tris-HCl, pH 8.0 and 25 mM EDTA, pH

8.0/0.5% SDS. Store at RT.
Proteinase K: Stored as 20 mg/ml aliquots at -20 C; Can be refrozen a few times.
Use Proteinase K at 0.3-0.5 mg/ml in digestion buffer
PCI: phenol/chloroform/isoamyl alcohol (from Ameresco)
DNA EXTRACTION FROM FROZEN

TISSUE SECTIONS
Tissue collection, storage, microdissection, sectioning: See separate protocol.
Tissue handling: Note that all fresh tissue should be handled as BioSafety Level 2
materials (wear gloves, lab coat, etc.).
DNA extraction: The following protocol is based on a standard phenol DNA extraction
protocol. Other protocols, and versions of this protocol, are also acceptable.
1. 1. Take pre-cut samples out of the –80°C freezer and thaw.
2. 2. Add 10 ml of PBS (Ca-Mg Free) in hood in BSL2 room to dissolve OCT. Invert
tube to make sure all of tissue is in the solution and not stuck on the tube walls.
3. 3. Spin down tissue 1400 rpm (500 x g) for 5min.
4. 4. Remove supernatant carefully watching the tissue pellet.
Note: Repeat steps 2-4 (with 5mls PBS) if it looks like there is significant “sticky”
OCT left in the tube. If you are repeating the PBS wash step you do not have to get
too close to the tissue pellet the first time.
5. 5. Resuspend pellet by vortexing. Add 950 ul digestion buffer (100 ul 10X PCR
buffer [100 mM TRIS, 15 mM MgCl2, 500 mM KCl), 5 ul 0.5% tween 20, 845 ul
H2O) and 30-50 ul of 20 mg/ml Proteinase K (PK, Sigma P2308).
Note: this volume should vary depending on the size of the tissue pellet. If the pellet
is bigger then add 2 ml total of buffer + PK.
6. 6. Resuspend pellet by vortexing, and pipeting up and down and place in a shaking
50°C water bath overnight. The next day make sure all the tissue has been digested.
If necessary, add more PK buffer and allow to digest for a few more hours.
7. 7. Split each tube into two 1.5ml tubes (500 ul per tube).
8. 8. Add 500ul (or equal volume) of room temp Phenol/Chloroform/Isoamyl

Alcohol (PCI from Amaresco) into each tube and vortex for 10sec.
Note: PCI is the lower organic layer.
9. 9. Centrifuge at 14000 rpm (max) for 2 min at room temp.

10. 10. Remove aqueous layer into a new tube and repeat the PCI extraction (steps 8-
10).
11. 11. Aliquot the aqueous phase into as few 1.5 ml epindorf tubes as possible.
Maximum volume per tube is 350 uls. Add 1/2 volume of 7.5 M ammonium acetate
and mix.
12. 12. Add 2.5 X 100% ethanol, mix by inversion. Leave at RT for 2 hrs, or ON at –
20°C.
13. 13. Centrifuge at 14000 rpm for 15 min @ 4°C.
14. 14. Decant supernatant immediately.***Note watch for the pellet***
15. 15. Wash pellet in 70 ul of 100% ethanol. Make sure you rinse sides, rim of tube.
Spin at 14000 rpm for 5 min and dump supernatant.
16. 16. Blot with Kimwipe. Air dry pellet.
17. 17. Add 20-50 ul of TE. The volume will depend on the pellet. (avg ~ 30 ul)
18. 18. Leave at RT for 2 hrs or ON.
19. 19. Place tubes into 65°C for 1 hour (to inactivate DNAse).
20. 20. Combine tubes. Rinse out “empty” tubes with 20 ul TE (the same 20 ul can be
used to rinse out all tubes).
21. 21. Measure DNA concentration using a fluorometer with a known standard DNA
solution. Very small amounts of DNA can be quantitated by TaqMan analysis with a
standard assay.
22. 22. Store DNA at 4°C for short periods, or colder for longer periods. Repeated
freezing and thawing may lead to shearing of DNA into smaller pieces.
A quick method to isolate plant genomic DNA

• Use from 0.01 - 0.1 gram plant material.
• Grind the plant material with liq. N2 in a mortar. We normally use
some alumina to crush hard tissue.
• Transfer the ground tissue to a eppendorf tube.
• Add 1 ml extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM
EDTA pH 8.0, 500 mM NaCl, + 0.07 % 2-mercaptoethanol). Mix
well.
• Add 130 ul 10% SDS, invert / shake the tube a few times. Incubate
at 65 C for 15 min.
• Add 300 ul 5M potassium acetate. Mix well. Keep the solution on
ice for i 30 min, (precipitation of proteins).
• Spin down the precipitations at 7000 rpm, 5 min. Transfer 300 ul of
the supernatant to a new eppendorf tube.
• Add 900 ul NaI (GeneClean II), + 20 ul "glass milk" (silica
particles) to the supernatant, (total volume of 1220 ul). Mix well
and incubate at room temp. for 5 min.
• Spin down the silica particles (glass milk), 5 sec., remove
supernatant. (The DNA in the solution will now hopefully be bound
to the silica particles).
• Wash the silica pellet with 800 ul wash solution (from the
GeneClean II kit).
• Repeat the wash two times.
• Dry the pellet (with bound DNA).
• Resuspend the pellet in 50 ul distilled water. Incubate at 50-65 C
for 5 minutes.
• Spin down pellet and transfer the eluted DNA to a new eppendorf
tube.
• At this point you should have enough DNA to run 10-20 PCR
reactions. Optional you can check 10 ul of the eluate on a agarose
gel. If you use 0.1 gram plant material you should be able to see the
DNA on the gel.
PLANT DNA EXTRACTION

A. thaliana has a very small haploid genome and this makes obtaining DNA
somewhat difficult. The most notable problem is that DNA is usually
contaminated with polysaccharide which inhibit restriction enzymes as well as
other DNA modifying enzymes. This problem is most easily solved by using
young plants which have not accumulated as much polysaccharide as older
plants. The best results are obtained with plants that are two to three weeks post
germinated.
1. Harvest plants using forceps - carefully remove any adhering soil

by hand.
2. Grind up the following in a mortar and pestle until no large pieces
of tissue remain:
0.5 - 1.5 g plants
0.5 g of glass beads (75-150 um) per gram of plants
3 ml proteinase K buffer (0.2 M Tris (pH 8.0), 0.1 M EDTA, 1%
Sarkosyl, 100 g/ml proteinase K)
3. Pour into 10 ml test tube. Incubate at 45-50 C for 1 hr.
4. Spin 10 min at top speed in table top centrifuge (~3000 rpm)
5. Decant supernatant to a fresh tube. Adjust volume to 3 ml with
proteinase K buffer (with or without proteinase K).
6. Add 6 ml 100% ethanol at room temperature. Invert to mix.
7. Spin 10K rpm for 15 min in SS34 rotor. Discard supernatant.
8. Resuspend pellet in 3 ml Tris-Cl (pH 8.0), 1 mM EDTA (TE).
Vortex to resuspend.
9. Extract with phenol, phenol:chloroform, chloroform.
10. Add 6 ml 100% ethanol. Invert to mix.
11. Spin 10K rpm for 15 min. in SS34 rotor. Discard supernatant.
Air-dry pellet briefly.
12. Resuspend in 4 ml TE. Vortex to resuspend.
13. Add 4,5 g CsCl, 400 l 10 mg/ml ethidium bromide. Mix.
14. Spin 53K rpm 16-20 hrs VTi65 20 C.
This protocol has been optimized for yield at the expense of high molecular
weight DNA. The nuclear DNA can be separated from plastid DNA by running
the gradients with Hoest dye, rather than ethidium bromide.
Plant DNA Extraction Protocol
Introduction
Plant materials are among the most difficult for high quality DNA
extractions. The key is to properly prepare the tissues for extraction.
In most cases this involves the use of liquid nitrogen flash freezing
followed by grinding the frozen tissue with a mortar and pestle. Liquid
nitrogen is difficult to handle and it is dangerous in an open
laboratory environment such as a classroom. For this reason we have
modified a very simple plant DNA extraction protocol to use fresh
tissue. We have also used tissue prepared in advance by dessication.
The protocols and results are presented here.
Reagents and Buffers

Extraction Buffer A (EBA): Per 100ml.
2% (w/v) hexadecyltrimethylammonium bromide (CTAB) 2.0g
100mM Tris (pH 8.0) (Use 1M stock) 10ml
20mM EDTA (Use 0.5M stock) 1ml
1.4M NaCl 8.2g
4% (w/v) polyvinylpyrrolidone (PVP) 4.0g
0.1% (w/v) ascorbic acid 0.1g
10mM _-mercaptoethanol (BME)* (Use 14.3M stock) 70μl
Extraction Buffer B (EBB): Per 100ml.
100mM Tris-HCl (pH 8.0) (Use 1M stock) 10ml
50mM EDTA (Use 0.5M stock) 2.5ml
100mM NaCl 0.6g
10mM _-mercaptoethanol (BME)* (Use 14.3M stock) 70μl
TE Buffer: Per 100ml.
10mM Tris (pH 8.0) (Use 1M stock) 1.0ml
1mM EDTA (Use 0.5M stock) 50μl
Other Required Reagents:
20% (w/v) sodium dodecyl sulphate (SDS)
5M potassium acetate (Stored at –20oC)
3M sodium acetate (pH 5.2)
70% ethanol (stored at -20oC)
Absolute isopropanol (stored at -20oC)
Extraction Protocol:
1. Weight out 0.3g of plant tissue
2. Place tissue on a clean glass slide. Chop the tissue into a paste
using a clean single
edge razor blade. (we have also modified a Dremel Roto-tool for use
as a simple
tissue homogenizer with good success)
3. Immediately transfer tissue to a 1.5ml microcentrifuge tube (use
Kontes #749520-
0090) and (optional) further grind tissue with a tube pestle (Kontes
#749521-
1590)
4. Once the sample is prepared add 300μl EBA, 900μl EBB, and 100μl
SDS.
5. Vortex and incubate at 65 oC for 10 min.
6. Place tube on ice and add 410μl cold potassium acetate. Mix by
inversion and
place tube back on ice for 3 min.
7. Centrifuge at 13,200rpm for 15 min. (If possible, use a refrigerated
microcentrifuge
set to 4 oC)
8. Transfer 1ml of the supernatant to a new 1.5ml microcentrifuge
tube, add 540μl
of ice cold absolute isopropanol, and incubate in ice for 20min.
9. Centrifuge at 10,200rpm for 10min. discard the supernatant. Wash
the pellet once
in 500μl 70% ethanol and let dry
10. Resuspend the dry pellet in 600μl of TE. Add 60μl 3M sodium
acetate (pH 5.2)
and 360μl ice cold absolute isopropanol. Incubate on ice for 20min.
11. Repeat Steps 9-11 twice.
12. Resuspend the pellet in 50μl TE and carry out agarose gel QC.
Agarose Gel QC:
1. Cast a 1.0% (w/v) regular agarose gel in 1x TBE
2. Place 5μl of extracted DNA and 5μl sterile water
in a 0.2ml microcentrifuge tube along with 2μl of
gel tracking dye.
Genomic
DNA
3. Run the gel for 20min. at 100v.
4. Stain gel and view result.
Cetyltrimethylammonium bromide (CTAB) Plant DNA Extraction

1. Grind 2 to 5 g of frozen leaves to a very fine powder with a liquid nitrogen-cooled
mortar and pestle.
2. Add 25 ml of CTAB Buffer and transfer to a 50 ml tube.
3. Incubate at 65°C for 20 min with occasional vigorous shaking.
4. Add 10 ml of Chloroform, shake well, and place on a tube inverter at room
temperature for 20 min.
5. Centrifuge at 1,000 X g for 5 min to resolve phases.
6. Transfer the aqueous phase to a fresh tube, add 17 ml of Isopropanol, mix, and
place on ice for 10 min.
7. Centrifuge at 1,000 X g for 5 min to collect the precipitate.
8. Discard the supernatant and dry the inside of the tube with a paper towel (do
not dry the pellet).
9. Add 4 ml of TE Buffer and dissolve the precipitate by gentle inversion.
10. Add 4 ml of 4 M Lithium Acetate and incubate on ice for 20 min.
11. Centrifuge at 1,000 X g for 10 min.
12. Transfer the supernatant to a fresh tube, add 16 ml of 100% Ethanol, and
incubate on ice for 20 min.
13. Centrifuge at 1,000 X g for 5 min to collect the precipitate.
14. Discard the supernatant and dry the inside of the tube with a paper towel (do
not dry the pellet).
15. Dissolve DNA in 900 μl of TE Buffer by gentle pipetting.
16. Add 100 μl of 3 M Sodium Acetate. Divide the sample evenly into two
microcentrifuge tubes.
17. Add an equal volume of Phenol to each tube, mix well, centrifuge to separate
the phases (1,000 X g), and save the aqueous phase (upper phase).
18. Add an equal volume of Phenol:Chloroform to each tube, mix well, centrifuge
to separate the phases (1,000 X g), and save the aqueous phase (upper phase).
19. Add an equal volume of Chloroform to each tube, mix well, centrifuge to
separate the phases (1,000 X g), and save the aqueous phase (upper phase).
20. Add 2 volumes of 100% Ethanol to each tube and incubate on ice for 5 min.
21. Centrifuge 5 min to collect the precipitate (approximately 5,000 X g).
MITOCHONDRIAL DNA ISOLATION
Description
MITOCHONDRIAL DNA ISOLATION
Procedure
1. Grind in mortar and pestle or Waring blender with 5-7 volumes

buffer A per g tissue. Use MCE at 350 l/L, and if necessary, with 5 ml 1
M DIECA/L.
2. Squeeze through cheesecloth, two layers of Miracloth.
3. 10 min at 1000 g
4. Decant supernatant and centrifuge 10 min at 15,900 g.
5. Resuspend each pellet in a few drops of buffer G with paint
brush; combine; bring to about 10 ml/50 g, 15 ml/75 g.
6. 10 min at 1000 g; pour off most; swirl pellet to remove fluffy
layer; combine.
7. Bring supernatant to 10 mM MgCl2 (100 l 1M/10 ml). Bring to
20 g DNase/ml (100 l 2mg/ml/10 ml).
8. 60 min. 4 C.
9. Underlay shelf buffer, 20 ml/10-15 ml; always use 20 ml or more.
10. 20 min at 12000 g.
11. Resuspend in small volume shelf buffer with brush; bring to about
10 ml/50-100 g.
12. 10 min at 15900 g.
13. Resuspend pellets in NN (lysis) buffer (4-5 ml/50-75 g).
14. Add SDS to 0.5% (250 l of 10%/5 ml NN). Swirl thoroughly.
15. Add proteinase K to 100 g/ml (25 l of 20 mg/ml/5 ml NN). Swirl
gently.
16. 60 min. 37 C.
17. Add equal volume of 3:1 water-saturated phenol, chloroform-
isoamyl alcohol mixture. Emulsify ca. 5 min.
18. 10 min at 7000 g.
19. Collect supernatant; repeat 17 and 18: 3 total extractions.
20. Final supernatant; add 0.1 volume 8 M Ammonium acetate; then
add 2 volumes of absolute ethanol.
21. 60 min, -80 C; 10 min at 8000-9000 g; drain; add equal volume
70% ethanol; let sit 10 min; 10 min at 8000-9000 g; drain dry. Vacuum
dry pellet, 30 min. Two small corex tubes are better than one 30 ml
Corex.
22. Add 100-500 l 0.1X NTE, 10 l RNase mixture. Typically use 500
l per 50 g tissue.
23. Hydrate 30 min., 37 C.
DNA EXTRACTION PROCEDURE –

GENERAL BACTERIA
1. Grow cells overnight in 500 ml broth medium.
2. Pellet cells by centrifugation, and resuspend in 5 ml 50 mM Tris
(pH 8.0), 50 mM EDTA.
3. Freeze cell suspension at -20C
4. Add 0.5 ml 250 mM Tris (pH 8.0), 10 mg/ml lysozyme to frozen
suspension, and let thaw at room temperature. When thawed, place on
ice for 45 min.
5. Add 1 ml 0.5% SDS, 50 mM Tris (pH 7.5), 0.4 M EDTA, 1
mg/ml proteinase K. Place in 50C water bath for 60 min.
6. Extract with 6 ml Tris-equilibrated phenol and centrifuge at
10,000X g for 15 min. Transfer top layer to new tube (avoid interface).
Re-do this step if necessary.
7. Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95%
ethanol (mix by inverting).
8. Spool out DNA and transfer to 5 ml 50 mM Tris (pH 7.5), 1 mM
EDTA, 200 g/ml RNase. Dissolve overnight by rocking at 4C.
9. Extract with equal volume chloroform (mix by inverting) and
centrifuge at 10,000X g for 5 min. Transfer top layer to a new tube.
10. Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95%
ethanol (mix by inverting).
11. Spool out DNA and dissolve in 2 ml 50 mM Tris (pH 7.5), 1 mM
EDTA.
12. Check purity of DNA by electrophoresis and spectrophotometric
analysis.
Preparation of Genomic DNA from Bacteria- using Phase

Lock GelTM
Reactives
TE buffer
10% (w/v) sodium dodecyl sulfate (SDS)
20 mg/ml proteinase K
phenol\chloroform (50:50)
isopropanol
70% etanol
3M sodium acetate pH 5.2
Phase Lock GelTM (Eppendorf-Brinkmann)
Procedure
1. Grow E. coli culture overnight in rich broth.

2. Transfer 2 ml to a 2-ml micro centrifuge tube and spin 2 min.
3. Decant the supernatant.
4. Drain well onto a Kimwipe.
5. Resuspend the pellet in 467 μl TE buffer by repeated pipetting.
6. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K, mix ,
and incubate 1 hr at 37 ° C.
7. Add an equal volume of phenol/chloroform and mix well but
very gently to avoid shearing the DNA by inverting the tube
until the phases are completely mixed. CAUTION: PHENOL
CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB
COAT AND KEEP TUBES CAPPED TIGHTLY.
8. Carefully transfer the DNA/phenol mixture into a Phase Lock
GelTM tube (green) and spin at 12,000 RPM for 10 min.
9. Transfer the upper aqueous phase to a new tube and add an
equal volume of phenol/chloroform.
10. Again mix well and transfer to a new Phase Lock GelTM
tube and spin 10 min.
11. Transfer the upper aqueous phase to a new tube.
12. Add 1/10 volume of sodium acetate. Mix.
13. Add 0.6 volumes of isopropanol and mix gently until the DNA
precipitates.
14. Spool DNA onto a glass rod (or Pasteur pipet with a heat-
sealed end).
15. Wash DNA by dipping end of rod into 1 ml of 70% ethanol
for 30 sec.
16. Resuspend DNA in at least 200 μl TE buffer. Complete
resuspension may take several days. Store DNA at 4 ° C short
term, -20 or -80 ° C long term.
17. After DNA has dissolved, determing the concentration by
measuring the absorbance at 260 nm.
Isolation and Quantification of Genomic

DNA from Mycobacterium tuberculosis
The following protocol describes a "DNA mini-prep" procedure (Part A) for the
isolation of chromosomal DNA from Mycobacterium tuberculosis and a procedure for
quantifying the amount isolated (Part B).
Part A. Isolation of Nucleic Acids

NOTE: CAUTION! STEPS 1-10 SHOULD BE PERFORMED USING
APPROPRIATE PROCEDURES FOR HANDLING MATERIAL POTENTIALLY
CONTAMINATED WITH MYCOBACTERIUM TUBERCULOSIS. ALSO,THE LIDS
OF THE EPPENDORF TUBES SHOULD BE CAREFULLY CLOSED AND
OPENED SO AS TO AVOID SPLASHES AND AEROSOL =46ORMATION.
• 1. Using a 1ml plastic, disposable pipet attached to a Pipet-Aid motorized

pipettor, add 1 ml sterile TE buffer to an L&J slant containing MTB colonies
selected for extraction of DNA. Using the end of the pipet, dislodge colonies
from surface of medium until all colonies are suspended in the 1 ml TE buffer.
Be careful not to disrupt the surface of the medium. Remove the suspended cells
in the TE buffer to a 1.5 ml, sterile, screw-capped microfuge tube and seal tube.
• 2. Place the sealed microfuge tube in an 80 deg. C oven for 60 min.
• 3. Centrifuge the Eppendorf tube for 5 min at room temperature using an
aerosol-containing microfuge operating at 9,000 rpm.
• 4. Carefully remove the supernatant using a disposable, cotton-plugged Pasteur
pipette. Discard the pipette in an appropriate manner.
• 5. To the remaining cell pellet, add 550 ml of Solution A. Thoroughly resuspend
the pellet using a vortex. Incubate the cell suspension at 37 deg. C for 1 hour.
• 6. To the cell suspension, now add 76 ml of Solution B. Thoroughly mix the
contents of the Eppendorf tube using a vortex. Incubate the cell suspension at 65
deg. C for 10 min.
• 7. Next, add 100 ml of 5 M NaCl to the cell suspension and thoroughly mix the
contents of the Eppendorf tube using a vortex. Then add 80 ml of CTAB/NaCl
and again thoroughly mix the contents of the Eppendorf tube using a vortex.
Incubate the resulting suspension at 65 deg. C for 10 min.
• 8. After the above incubation step, add 700 ml of chloroform/isoamyl alcohol.
Thoroughly mix the contents of the Eppendorf tube at least 15 sec using a
vortex. Then, centrifuge the Eppendorf tube for 5 min at room temperature using
a microfuge operating at 14,000 rpm (@15,300 x g).
• 9. Using a disposable pipette, remove the upper aqueous layer (without
disturbing or carrying over any of the white middle layer) to a second 1.5 ml
Eppendorf tube. Fill the remaining volume of the Eppendorf tube with
isopropanol, seal the tube, and invert it several times to mix the contents.
Incubate the tube at -20 deg. C at least 30 min.
NOTE: After adding the isopropanol, the Eppendorf tubes can be stored at -20
deg. C until transported to the general microbiology laboratory or they can be
immediately taken to the General microbiology laboratory for theincubation at
-20 deg. C and subsequent handling.
• 10. Collect the nucleic acids by centrifugation for 30 min using a microfuge
operating at 14,000 rpm (@15,300 x g). Gently drain off the supernatant, then
carefully add approximately 1 ml of cold 70% (v/v) ethanol. Again, collect the
nucleic acids by centrifugation for 15 min using a microfuge operating at 14,000
rpm (@15,300 x g).
• 11. Carefully drain off the supernatant and evaporate the remaining ethanol
using the Speed-Vac Concentrator for 30 min.
• 12. Dissolve the nucleic acid pellet in 50 ml TE Buffer. Be sure to dissolve any
of the precipitate adhering to the "spine" of the Eppendorf tube by washing it
with the TE buffer.
• 13. Optional Step: To remove contaminating RNA from the preparation, add 1
ml of RNase to the nucleic acid solution. Incubate the tube at 37 deg. C for 30
min.
NOTE: The nucleic acid solution should be stored at -20 deg. C when not in =
use.
Part B. Determination of DNA Concentration
• 1. Turn on the Hoeffer DNA Fluorimeter and allow it to warm up at least 30

min. (NOTE: If the sample was stored frozen, allow it to thaw at room
temperature. Once thawed, keep the sample on ice until it is processed.)
• 2. Prepare a 25 ng/ml solution of Lambda Phage DNA to be used as a standard
for determining the concentration of DNA derived from the various M.
tuberculosis strains. This is done by diluting 20 ml of Lambda Phage DNA (at a
concentration of 0.25 mg/ml) with 180 ml of sterile, distilled-deionized water.
This standard solution can now be stored frozen at -20 deg. C until needed. Also,
it can be repeatedly frozen/thawed and kept on ice during use.
• 3. Pass 100 ml of 1X TNE buffer through a 0.22 mm filter to remove any
particulate matter. To this buffer, add 10 ml of Hoescht dye 33257 (1
mg/ml)=DD to obtain a final dye concentration of 100 ng/ml.
• 4. Place 2.00 ml of the dye/buffer solution (prepared in Step 3) in the cuvette.
Zero the fluorimeter.
• 5. Remove the cuvette, add exactly 1.0 ml of the Lambda Phage DNA standard,
mix by inversion using parafilm to cover the cuvette opening, replace the cuvette
in the machine, and record the absorbance.
• 6. Repeat Step 5 using various volumes of Lambda Phage DNA standard added
to the dye/buffer solution up to a cumulative added volume of 20 ml. Be sure to
record the absorbance for the total volume (in ml) of Lambda Phage DNA
added.
• 7. Thoroughly wash the cuvette with the dye/buffer solution prepared in Step 3.
Shake as much excess dye/buffer solution from the cuvette as possible.
• 8. Place 2.00 ml of the dye/buffer solution (prepared in Step 3) in the cuvette,
then add exactly 1.0 ml of the DNA to be quantitated, mix by inversion using
parafilm to cover the cuvette opening, replace the cuvette in the machine, and
record the absorbance. If the absorbance falls above the upper reading of the last
standard measured, prepare an appropriate dilution of the DNA sample and
repeat Steps 7 and 8. If the absorbance falls below the lower reading of the first
standard measured, add one to several more ml of the sample until a reading is
obtained. Be sure to record the volume of DNA sample used to obtain a
particular absorbance reading.
• 9. Repeat Steps 7 and 8 for each DNA sample to be quantitated.
• 10. Using the data from Steps 5 and 6, prepare a standard curve to determine the
concentrations (in mg/ml) from the absorbance readings from the DNA samples
measured in Steps 7 through 9.

V. Working with DNA
A. Storage
The following properties of reagents and conditions are important

considerations in processing and storing DNA and RNA. Heavy metals
promote phosphodiester breakage. EDTA is an excellent heavy metal
chelator. Free radicals are formed from chemical breakdown and radiation
and they cause phosphodiester breakage. UV light at 260 nm causes a
variety of lesions, including thymine dimers and cross-link. Biological
activity is rapidly lost. 320 nm irradiation can also cause cross-link, but
less efficiently. Ethidium bromide causes photo oxidation of DNA with
visible light and molecular oxygen. Oxidation products can cause
phosphodiester breakage. If no heavy metal are present, ethanol does not
damage DNA. Nucleases are found on human skin; therefore, avoid direct
or indirect contact between nucleic acids and fingers. Most DNases are not
very stable; however, many RNases are very stable and can adsorb to glass
or plastic and remain active. 5 E C is one of the best and simplest
conditions for storing DNA. -20 deg C: this temperature causes extensive
single and double strand breaks. -70 E C is probable excellent for long-
term storage. For long-term storage of DNA, it is best to store in high salt (
>1M) in the presence of high EDTA ( >10mM) at pH 8.5. Storage of DNA in
buoyant CsCl with ethidium bromide in the dark at 5 E C is excellent. There
is about one phosphodiester break per 200 kb of DNA per year. Storage of
λ DNA in the phage is better than storing the pure DNA. [ ref: Davis, R.W.,
D. Botstein and J.R. Roth, A Manual for Genetic Engineering: Advanced
Bacterial Genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor,
N.Y. 1980.]
B. Purification
To remove protein from nucleic acid solutions:
Treat with proteolytic enzyme, e.g., pronase, proteinase K

Purify on a silica-based column such as a Qiagen PCR Prep Column
CsCl/ethidium bromide density gradient
Phenol Extract. The simplest method for purifying DNA is to extract with
phenol or phenol:chloroform and then chloroform. The phenol
denatures proteins and the final extraction with chloroform
removes traces of phenol
Purify on silica-based column such as Qiagen Brand columns
(http://www.qiagen.com)
C. Quantitation .
Spectrophotometric. For pure solutions of DNA, the simplest method of

quantitation is reading the absorbance at 260 nm where an OD of 1
in a 1 cm path length = 50 μ g/ml for double-stranded DNA, 40 μ
g/ml for single-stranded DNA and RNA and 20-33 μ g/ml for
oligonucleotides. An absorbance ratio of 260 nm and 280 nm gives
an estimate of the purity of the solution. Pure DNA and RNA
solutions have OD 260/OD 280 values of 1.8 and 2.0, respectively.
This method is not useful for small quantities of DNA or RNA (<1 μ
g/ml).
Ethidium bromide fluorescence. The amount of DNA is a solution is
proportional to the fluorescence emitted by ethidium bromide in
that solution. Dilutions of an unknown DNA in the presence of 2 μ
g/ml ethidium bromide are compared to dilutions of a known
amount of a standard DNA solutions spotted on an agarose gel or
Saran Wrap or electrophoresed in an agarose gel.
D. Concentration
Precipitation with ethanol. DNA and RNA solutions are concentrated with
ethanol as follows: The volume of DNA is measured and the monovalent
cation concentration is adjusted. The final concentration should be 2-2.5M
for ammonium acetate, 0.3M for sodium acetate, 0.2M for sodium chloride
and 0.8M for lithium chloride. The ion used often depends on the volume of
DNA and on the subsequent manipulations; for example, sodium acetate
inhibits Klenow, ammonium ions inhibit T4 polynucleotide kinase, and
chloride ions inhibit RNA-dependent DNA polymerases. The addition of
MgCl 2 to a final concentration of 10mM assists in the precipitation of small
DNA fragments and oligonucleotides. Following addition of the monovalent
cations, 2-2.5 volumes of ethanol are added, mixed well, and stored on ice
or at -20 E C for 20 min to 1 hour. The DNA is recovered by centrifugation
in a microfuge for 10 min (room temperature is okay). The supernatant is
carefully decanted making certain that the DNA pellet, if visible, is not
discarded (often the pellet is not visible until it is dry). To remove salts,
the pellet is washed with 0.5-1.0 ml of 70% ethanol, spun again, the
supernatant decanted, and the pellet dried. Ammonium acetate is very
soluble in ethanol and is effectively removed by a 70% wash. Sodium
acetate and sodium chloride are less effectively removed. For fast drying,
the pellet can spun briefly in a Speedvac, although the method is not
recommended for many DNA preparations as DNA that has been over dried
is difficult to resuspend and also tends to denature small fragments of
DNA. Isopropanol is also used to precipitate DNA but it tends to
coprecipitate salts and is harder to evaporate since it is less volatile.
However, less isopropanol is required than ethanol to precipitate DNA and
it is sometimes used when volumes must be kept to a minimum, e.g., in
large scale plasmid preps.
E. Restriction Enzymes
Restriction and DNA modifying enzymes are stored at -20 deg C in a non-
frost free freezer, typically in 50% glycerol. The enzymes are stored in an
insulated cooler which will keep the enzymes at -20 deg C for some period
of time. The tubes should never be allowed to reach room temperature and
gloves should be worn when handling as fingers contain nucleases. Always
use a new, sterile pipet tip every time you use a restriction enzyme. Also,
the volume of the enzyme should be less than 1/10 of the final volume of
the reaction mixture.

IV. Equipment
A. General Comments
It is to everyone's advantage to keep the equipment in good working

condition. As a rule of thumb, don't use anything unless you have been
instructed in the proper use. This is true not only for equipment in the lab
but also departmental equipment. Report any malfunction immediately.
Rinse out all centrifuge rotors after use and in particular if anything spills.
Please do not waste supplies - use only what you need. If the supply is
running low, please notify either the instructor/lab managerbefore the
supply is completely exhausted. Occasionally, it is necessary to borrow a
reagent or a piece of equipment from another lab. Except in an emergency,
notify the instructor.
B. Micropipettors
Most of the experiments you will conduct in this laboratory will depend on
your ability to accurately measure volumes of solutions using
micropipettors. The accuracy of your pipetting can only be as accurate as
your pipettor and several steps should be taken to insure that your
pipettes are accurate and are maintained in good working order. Each pair
of students will be assigned a set of pipettors and upon receipt, they
should be labeled with the students' name. They should then be checked
for accuracy following the instructions given by the instructor. If they need
to be recalibrated, do so. We have two different types of pipettors, Rainin
pipetmen and Oxford benchmates. Since the pipettors will use different
pipet tips, make sure that the pipet tip you are using is designed for your
pipettor. DO NOT DROP IT ON THE FLOOR. If you suspect that something is
wrong with your pipettor, first check the calibration to see if your
suspicions were correct, then notify the instructor.
C. Using a pH Meter
Biological functions are very sensitive to changes in pH and hence, buffers

are used to stabilize the pH. A pH meter is an instrument that measures
the potential difference between a reference electrode and a glass
electrode, often combined into one combination electrode. The reference
electrode is often AgCl 2. An accurate pH reading depends on
standardization, the degree of static charge, and the temperature of the
solution.
Operation of Orion PerpHecT pH Meter
Expose hole on side of electrode by sliding the collar down. Make sure
there is sufficient electrode filling solution in the electrode (it
should be up to the hole). If not, fill with ROSS filling solution only
(Do not use any filling solution containing silver (Ag).
Ensure that sample to be pHed is at room temperature and is stirring
gently on the stir plate.
Calibrate the pH meter with the two solutions that bracket the target pH -
4 and 7 or 7 and 10 as follows:
Press the CAL key to initialize the calibration sequence. The last calibration
range will be displayed (e.g. 7-4). Press YES to accept or use the
scroll keys to select a different range. Press YES to accept.
The number 7 will light up on the left hand side of the screen indicating
that the meter is ready to accept the pH 7 standard buffer. Rinse
off electrode and place in fresh pH 7 standard buffer solution. The
READY light will come on when the value has stabilized. Press YES
to accept the value.
The number 4 (or 10) will light up next indicating that the meter is ready
to accept the pH 4 (or 10) standard buffer solution. Rinse off
electrode and place in fresh pH 4 standard buffer solution. The
READY light will come on when the value has stabilized. Press YES
to accept the value.
SLP will be displayed. The meter will then go MEASURE mode.
Rinse electrode and place into sample. The READY light is displayed when
signal is stable.
D. Autoclave Operating Procedures
Place all material to be autoclaved in a autoclavable tray. All items should

have indicator tape. Separate liquids from solids and autoclave separately.
Make sure lids on all bottle are loose. Do not crowd large number of items
in tray- in order for all items to reach the appropriate temperature, one
must allow sufficient air/steam circulation.
Make sure chamber pressure is at 0 before opening the door.

Place items to be autoclaved in the autoclave and close the door. Some
autoclaves require that you also lock the door after it's closed.
Set time - typically 20 minutes.
Temperature should be set at 121 deg C already, but double-check and
change if necessary.
Set cycle: If liquid, set "liquid cycle" or "slow exhaust". If dry, set "dry
cycle" or "fast exhaust" + dry time.
Start the cycle. On some autoclaves, the cycle starts automatically at step
5. On others, turn to "sterilize".
At the end of the cycle, check that: a. the chamber pressure is at 0; b. the
temp is <100 deg C
Open door.
Remove contents using gloves and immediately tighten all caps.
E. Operating Instructions for Spectrophotometer - Pharmacia Ultraspec
To measure the absorbance of a solution in the short-wave range (<300

nM) use the quartz cuvettes. Disposable plastic cuvettes are
available for reading in the visible range.
Turn the spectrophotometer on - the switch is on the right in the back.
Allow the instrument to calibrate. Do not open the chamber during this
time. The deuterium lamp is OFF by default. To read absorbance in
the UV range, turn the deuterium lamp on as follows after the
machine has completed its calibration: Depress the function key
until Fn5 is displayed. Press the mode key until d2on is displayed.
Press enter. For best accuracy, the deuterium lamp should be
warmed up for 20 minutes.
Press the function key until Fn0 is displayed. Press enter. Using the up or
down arrow keys, enter in the desired wavelength.
Prepare a reference cuvette containing the same diluent as your
sample.Prepare your sample.
Place the reference cuvette in cell #1 and place your samples in cells #2-6.
Press the cell key until cell #1 is in position. Press the Set Reference key to
blank against the appropriate buffer. Press the cell key to advance
to read the next sample.

VI. Sterile Technique
All media, including plates, liquid media and top agar must be autoclaved
immediately after it is prepared. It is best to prepare media in
several small bottles, only opening one at a time. Check the bottle
for contamination before you use it by gently swirling it and looking
for cloudy material in the center. Always grow up a small amount of
broth alone when growing cells overnight. A small amount of
contamination is not always evident until the media is incubated at
37 deg C.
Use a flame on inoculating loops and on the lips of media bottles before
and after pipetting from them. Never leave a media or agar bottle
open on the bench and don's take an individually-wrapped pipet
out of its protective wrapper until you are ready to use it (i.e.,
don't walk across the room with an unwrapped pipet). Always use
a fresh, sterile pipet or pipet tip when pipetting culture media, and
never go back into a media bottle or cell culture with a used pipet.
To prevent wide-scale, untraceable contamination, each person should
have his own stock of liquid culture media, top agar, plates, 100%
glycerol, glycerol stocks of cells, etc. and don't share.
Overnight cultures should be grown only from a single colony on a fresh
plate or from a previously-tested glycerol stock that was grown
from a single colony. To prepare an overnight culture from a
glycerol stock, take an individually-wrapped 1-ml pipet and a
culture tube of media to the -80 deg C freezer. Quickly remove the
cap from the freezer vial containing the glycerol stock, scrap a
small amount of ice from the surface of the culture, replace the cap
on the freezer vial, and place the pipet into the culture tube.
Sufficient numbers of bacteria are present in the ice in order for
the culture to grow to saturation in 16 hours. Never let the glycerol
stock thaw.
Think about what you are doing. The best defense is common sense.
References
1. Boom,R., C.J.A. Sol , M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim van
Dillen and J. van *der Noordaa. 1989. Rapid and Simple Method for Purification
of Nucleic Acids. J. Clin. Microbiol. 28:495-503.
2. Bush,C. and M. Harvey. 1991. Rapid isolation of genomic DNA from whole
blood to borosilicate particles. Clin. Chem. 37:1060.
3. Marko,M.A., R. Chipperfield and H.C. Birnboim. 1982. A procedure for the
large-scale isolation of highly purified plasmid DNA using alkaline extraction
and binding to glass powder. Anal. Biochem. 121:382-387.
4. Vogelstein,B. and D. Gillespie. 1979. Preparative and analytical purification of
DNA from agarose. Proc.Natl.Acad.Sci USA. 76:615-619.
5. The following set of protocols were contributed by various members of our lab
(past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross
Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling
Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and
Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria
Murphy, who contributed many of the ES cell protocols. Sections dealing with
Photomicroscopy, Polyclonal and Monoclonal Antibody Production were
provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments
in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998
6. Maxim V.Myakishev*, George I.Kapanadze, Gadji O.Shaikhayev, Svetlana
auMalahova, Georgii P.Georgiev and David R.Beritashvili "Use of perchlorate
precipitation to improve plasmid isolation" Institute of Gene Biology, Moscow,
1995
7. Author: Laura-Lee Boodram, Laura-Lee Boodram, Department of Life Sciences, The University of the
West Indies, Date Added: Mon May 17 2004, Date Modified: Tue May 25 2004
8. Helms, C. Salting out Procedure for Human DNA extraction. In The Donis-
Keller Lab - Lab Manual Homepage [online]. 24 April 1990. [cited 19
November 2002; 11:09 EST]. Available from:
http://hdklab.wustl.edu/lab_manual/dna/dna2.html.
9. Epplen, J.E., and T. Lubjuhn. 1999. DNA profiling and DNA fingerprinting.
Birhkhauser Verlag, Berlin. p.55.
10. M. Ralser, R. Querfurth, H.J. Warnatz, H. Lehrach, M.L. Yaspo and S. Krobitsch An
efficient and economic enhancer mix for PCR BBRC, 2006.
doi:10.1016/j.bbrc.2006.06.151
11. S.A. Miller, Dykes, D. D., and H. F. Polesky . (1988). " A simple salting out
procedure for extracting DNA from human nucleated cells" Nucleic Acids
Research 16: 1215.
12. Laird PW, Nucleic Acids Research 1991,19:4293
13. Meyerowitz et. al. (~1987ish)
14. Protocol modified from Keb-Llanes et al. Plant Molecular Biology
Reporter 20:299a-299e. 2002.
15. Written by Chet Cooper, Ph.D., January 14, 1993
Appendix: Reagents/Solutions
Tween/albumin medium
1. Dissolve the following components in 995 ml of distilled-deionized water:
Component Amount Added:
 KH2PO4 1.5 g
 Na2HPO4 . 2H20 1.9 g
 Na3citrate . 2H2O 0.4 g
 MgSO4 . 7H2O 0.05g
 CaCl2 . 2H2O (0.5 g/l) 1 ml
 ZnSO4 . 7H2O (0.5 g/l) 2 ml
 CuSO4 . 5H2O (0.5 g/l) 2 ml
 Sodium glutamate 0.5 g
 (NH4)2SO4 0.5 g
 Glycerol 2 ml
 NH4Fe(III)citrate (10 g/l) 1 ml
 Pyridoxine HCl (1 mg/ml) 1 ml
 Biotin (0.1 mg/ml) 5 ml
 Tween 80 (50 ml/l) 10 ml
 Albumin-glucose solution* 100 ml
*This solution is made by dissolving 5 g of bovine albumin and 5 g of

glucose in 100 ml of distilled-deoinized wate and adding 0.1 ml of a 100
g/l sucrose solution.
2. Filter sterilize the medium using a 0.22 mm membrane filter.
Solution A
To 5.50 ml of TE Buffer (see below), add 10 mg of lysozyme. Mix thoroughly
using a vortex. (NOTE: Not all the lysozyme will dissolve in the buffer.)
Dispense 600 ml of the solution into 1.5 ml Eppendorf tubes and store frozen at
-20 deg. C. Use one tube per strain being sure to mix the solution thoroughly
prior to using. Discard the remaining solution.
Solution B
• 1. Prepare a 10% sodium lauryl sulfate (SDS; also known as sodium dodecyl
sulfate) solution by dissolving 50 g SDS in 400 ml of distilled-deio nized wate.
The solution will be cloudy, but adjust the pH to 7.2 using HCl. Bring the final
volume to 500 ml with distilled-deionized water. This solution can be stored at
room temperature without sterilization. (If the solution remains cloudy or
becomes cloudy in the future, warm it to 50-65=B0C to dissolve the SDS before
dispensing.)
• 2. Prepare a 10 mg/ml solution of proteinase K in distilled-deionized water.
Dispense aliquots to 1.5 ml Eppendorf tubes and store at -20 deg. C. This
solution can undergo several freeze-thaw cycles before the enzyme begins to
deteriorate.
• 3. Solution B is made by mixing 700 ml of the 10% SDS solution with 60 ml of
the proteinase K solution in a 1.5 ml Eppendorf tube. This solution can be stored
at -20 deg. C and can undergo several freeze-thaw cycles before the enzyme
begins to deteriorate.
5 M NaCl
Dissolve 29.22 g of NaCl in 100 ml of distilled-deionized water. Store this
solution at room temperature.
CTAB/NaCl
Dissolve 4.1 g of NaCl in 80 ml of distilled-deionized water using a magentic
stirrer and stir bar. While stirring, add 10 g CTAB
(Hexzadecyltrimethylammonium bromide). If needed, dissolve the CTAB by
heating the solution to 65 deg. C. Allow the solution to cool to room
temperature. Adjust the final volume to 100 ml with distilled-deionized water.
Chloroform/Isoamyl alcohol
Mix 96 ml chloroform with 4 ml isoamyly alcohol. Store this reagent at 4 deg.
C.
TE Buffer, pH 8.0
• 1. Make a 1.0 M Tris buffer solution by adding 121.1 g of Trizma base (Tris) to
1.0 l of distilled-deionized water. Adjust the pH to 8.0 using HCl or NaOH. This
solution can be sterilized by autoclaving and stored at room tempeature.
• 2. Make a 0.5 M EDTA solution by adding 186.1 g of Na2EDTA to 800 ml of
distilled-deionized water. Adjust the pH to 8.0 by adding approximately 20 g
NaOH pellets and mixing thoroughly. Make the final pH adjustments using a
NaOH solution. Bring the final volume to 1.0 l with distilled-deionized water.
This solution can be sterilized by autoclaving and stored at room temperature.
• 3. To 800 ml distilled-deionized water, add 10.0 ml of 1.0 M Tris, pH 8.0 and
2.0 ml 0.5 M EDTA, pH 8.0. Bring the total volume to 1.0 l with distilled-
deionized water. If necessary, adjust the pH to 8.0 using HCl or NaOH. This
solution can be sterilized by autoclaving and stored at room temperature. The
final concentrations of the components are 10 mM Tris and 1 mM EDTA.
RNase
Prepare DNase-free RNase by dissolving RNase A in a screw-cap tube at a
concentration of 2 mg/ml in a buffer containing 10 mM Tris (pH 7.6) and 15
mM NaCl. Place the tube in a boiling water bath for 15 min, then allow it to
slowly cool to room temperature. Dispense into small aliquots and store frozen
at -20 deg C. Thaw just prior to use. A single aliquot of RNase can be used
multiple times if refrozen and stored at -20 deg. C.
1X TNE Buffer
• 1. Prepare a 1.0 M Tris (pH 8.0) and a 0.5 M EDTA (pH 8.0) solutions as
described for the TE buffer formulation given above.
• 2. To 900 ml distilled-deionized water, add 100 ml of 1.0 M Tris, pH 8.0 and 20
ml 0.5 M EDTA, pH 8.0. Dissolve 58.4 g NaCl in this solution and then bring
the total volume to 1.0 l with distilled-deionized water. If necessary, adjust the
pH to 8.0 using HCl or NaOH. This solution is considered to be 10X TNE and it
can be stored at room temperature.
• 3. Prepare a 1X TNE buffer solution by diluting 10 ml 10X TNE to a final
volume of 100 ml using distilled-deionized water.
Warning!!!!!!!! xylene and ethanol are flammable and

heating flammable solvents is dangerous. The
flashpoint of xylene is 27oC. Ethanol has a
flashpoint of 12 oC . (Flashpoint is the minimum
temperature at which a liquid gives off a vapor in
sufficient concentration to ignite). Because of the
inherent dangers of heating flammable solvents: 1.)
Always conduct experiments in a chemical fume
hood 2.) Ensure that the hood is free from all
potential sources of ignition including electrical
outlets, power strips, and electrical equipment,
flames ect. 3.) Remove all other flammable solvents
and oxidizers from the hood before beginning. 4.)
Wear a lab coat, chemically resistant gloves, and a
face shield to protect against splashing. 5.) Heating
solvents in closed tubes can lead to pressure build
up inside the tubes causing the cap to pop open and
potential splashing. The cap should be opened to
relieve pressure before handling or kept open
during the 15 minute incubation.

General Laboratory Procedures

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

General Laboratory Procedures

Uploaded by

Copyright:

Available Formats

General Laboratory Procedures, Equipment Use, and

Arturo Duarte Ortuño

A number of chemicals used in any molecular biology laboratory are

The following chemicals are particularly noteworthy:

Phenol - can cause severe burns

The voltages used for electrophoresis are sufficient to cause electrocution.

General Laboratory Procedures, Equipment Use, and

A. Calculation of Molar, % and "X" Solutions .

1. A molar solution is one in which 1 liter of solution contains the number

2. Percent solutions.Percentage (w/v) = weight (g) in 100 ml of solution;

3. "X" Solutions. Many enzyme buffers are prepared as concentrated

B. Preparation of Working Solutions from Concentrated Stock Solutions .

C. Steps in Solution Preparation:

Refer to a laboratory reference manual for any specific instructions on

D. Glassware and Plastic Ware .

Glassware should be rinsed with distilled water and autoclaved or baked at

III. Methods for DNA isolation

3. Resuspend the cell pellets in a total of 70 ml of GET/Lysozyme solution (35 ml for

For cesium chloride-gradient purification:

9a. Dissolve the pellets in a combined total of 32 ml of 100:10 TE buffer, 5 ml of 5

15a. Resuspend the DNA in 10:0.1 TE buffer.

For diatomaceous earth-based purification:

10b. Decant the supernatant, resuspend each pellet in 40 ml of diatomaceous earth-wash

15b. Combine the DNA-containing supernatants and precipitate the DNA in 35 ml

B. Midiprep double-stranded DNA isolation

1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 12 X 75 mm

3. Resuspend the cell pellets in 2 ml of GET/Lysozyme solution, add 4 ml of alkaline

4. Add 4 ml of 3M NaOAc, pH 4.8, gently mix by swirling, and incubate in an ice-water

6. Decant the supernatant to a 50 ml polypropylene centrifuge tube, add 20 ul of a 20

7. Add 7 ml (equal volume) of de-fined diatomaceous earth in guanidine-HCl (20

8. Decant the supernatant, resuspend in 7 ml of diatomaceous earth-wash buffer, and

9. Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as above.

10. Decant the supernatant and dry in a vacuum oven.

C. Miniprep double-stranded DNA isolation

Since highly supercoiled DNA is desired for double-stranded DNA sequencing, a

3. Resuspend the cell pellets in 0.2 ml of TE-RNase solution (50:10 TE buffer

4. Add 0.2 ml of 3M NaOAc, pH 4.8, gently mix by swirling, transfer to 1.5 ml

6. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube, incubate in an ice-

For standard alkaline lysis purification:

For diatomaceous earth-base purification:

D. Large scale M13RF isolation (9)

1. Prepare an early log phase culture of JM101 by inoculating an Ehrlenmeyer flask

8. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in the Sorvall OTD-75B

12. Resuspend the DNA in 10:0.1 TE buffer.

E. Single-stranded M13 DNA isolation using phenol

3. Pipette the top 1 ml of supernatant to a fresh 1.5 ml microcentrifuge tube containing

5. Resuspend the pellet in 100 ul of 10 mM Tris-HCl, pH 7.6 by vortexing, and add 50

7. Resuspend the dried DNA in 6 ul of 10:0.1 TE for use in single-stranded

F. Biomek-automated modified-Eperon isolation

1. Prepare an early log phase JM101 culture in 50 ml of 2xTY, as above.

2. Using sterile toothpicks, transfer individual M13 plaques into 12 X 75 mm Falcon

3. Separate the bacterial cells from the viral-containing supernatant by centrifugation at

12. Resuspend the DNA templates in 20 ul of 10:0.1 TE buffer.

G. 96 well double-stranded template isolation

Manual Double stranded isolation method

The following is a manual, 96 well, double stranded sequencing template isolation

The following is an automated, 96 well, double stranded sequencing template isolation

5. Press ENTER to continue with the program.

H. Genomic DNA isolation from blood

Protocol (updated 10/30/98)

1. Blood samples typically were obtained as 1 ml of whole blood stored in EDTA

6. Add 120 ul phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge