Professional Documents
Culture Documents
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PCR EXPERIMENTAL SUCCESS GUIDELINES
Please refer to the Appendices section for a summary of important hints and re-
minders which will help maximize successful implementation of this experiment.
This experiment has three modules:
I. Isolation of DNA
II. PCR Amplication of DNA
III. Separation of PCR Reactions by Electrophoresis
MICROPIPETTING BASICS AND PRACTICE GEL LOADING
Accurate pipeting is critical for maximizing successful experiment results. ED-
VOTEK Series 300 experiments are designed for students who have had previous
experience with agarose gel electrophoresis and micropipeting techniques. If
your students are unfamiliar with using micropipets, EDVOTEK highly recom-
mends that students perform Experiment # S-44, Micropipetting Basics, or other
Series 100 or 200 electrophoresis experiment prior to conducting this advanced
level experiment.
APPROXIMATE TIME REQUIREMENTS
1. The PCR step (35 cycles) will take about 150-180 minutes or can be processed
overnight and held at 4C.
2. The experiment can be temporarily stopped after the completion of Module
1 or Module II and later resumed. Experimental results will not be compro-
mised if instructions are followed as noted under the heading Optional
Stopping Point.
3. Whether you choose to prepare the gel(s) in
advance or have the students prepare their
own, allow approximately 30-40 minutes
for this procedure. Generally, 20 minutes
of this time is required for gel solidication.
See section Options for Preparing Agarose
Gels below.
4. The approximate time for electrophore-
sis will vary from 40 minutes to 3.5 hours.
Generally, the higher the voltage applied,
the faster the samples migrate. However,
depending upon the apparatus congura-
tion and the distance between the two
electrodes, individual electrophoresis units
will separate DNA at different rates. Follow
manufacturer's recommendations. Time
and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.
Time and Voltage Guidelines
2% UltraSpec-Agarose Gel
Time Volts
125
70
50
40 min
2.0 hrs
3.0 hrs
Approx. migration
distance of
tracking dye
Gel Size (cm)
Volume
7 x 7
25 ml
7 x 14
50 ml
125
70
50
60 min
2.5 hrs
3.5 hrs
4.5 cm
6.0 cm
Table
C
Notes to the Instructor
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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Experiment
Identication of Foodstuffs
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OPTIONS FOR PREPARING AGAROSE GELS
This experiment is designed for DNA staining after electrophoresis with InstaStain
Ethidium Bromide. There are several options for preparing agarose gels for the
experiment.
1. Individual Gel Casting:
Each student lab group can be responsible for casting their own individual
gel prior to conducting the experiment.
2. Preparing Gels in Advance:
Gels may be prepared ahead and stored for later use. Solidied gels can be
stored under buffer in the refrigerator for up to 2 weeks.
Do not store gels at -20C. Freezing will destroy the gels.
Gels that have been removed from their trays for storage, should be "an-
chored" back to the tray with a few drops of hot, molten agarose before
placing the gels into the apparatus for electrophoresis. This will prevent the
gels from sliding around in the trays and the chambers.
3. Batch Gel Preparation:
A batch of agarose gel can be prepared for sharing by the class. To save
time, a larger quantity of UltraSpec-Agarose can be prepared for sharing by
the class. See instructions for "Batch Gel Preparation".
GEL CONCENTRATION AND VOLUME
The gel concentration required for this experiment is 2.0%. Prepare gels accord-
ing to Table A.1 or A.2 in Appendix D.
Notes to the Instructor
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GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS
After electrophoresis, the agarose gels require staining in order to visualize the
separated DNA samples. This experiment features a proprietary stain called
InstaStain.
InstaStain EtBr (Appendix F)
Optimal visualization of PCR products on gels of 1.0% or higher concentration is
obtained by staining with InstaStain Ethidium Bromide (InstaStain EtBr) cards.
Exercise caution when using Ethidium Bromide, which is a listed mutagen. Dispos-
al of the InstaStain EtBr cards, which contain only a few micrograms of ethidi-
um bromide, is minimal compared to the large volume of liquid waste generated
by traditional ethidium bromide staining procedures. Disposal of InstaStain
cards and gels should follow institutional guidelines for chemical waste.
InstaStain Blue: One-step Staining and Destaining (Appendix G)
InstaStain Blue can be used as an alternative for staining gels in this experi-
ment. However, InstaStain Blue is less sensitive than InstaStain EtBr and will
yield variable results.
Agarose gels can be stained and destained in one easy step, which can be com-
pleted in approximately 3 hours, or can be left in liquid overnight. For the best
photographic results, leave the gel in liquid overnight. This will allow the stained
gel to equilibrate in the destaining solution, resulting in dark blue DNA bands
contrasting against a uniformly light blue background.
Gels stained with InstaStain Blue may be stored in the refrigerator for several
weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT
FREEZE AGAROSE GELS! Used InstaStain Blue cards and destained gels can be
discarded in solid waste disposal. Destaining solutions can be disposed down the
drain.
PHOTODOCUMENTATION OF DNA (OPTIONAL)
There are many different photodocumentation systems available, including digi-
tal systems that are interfaced directly with computers. Specic instructions will
vary depending upon the type of photodocumentation system you are using.
Notes to the Instructor
21
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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Experiment
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from Genetically Modied Organisms
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Pre-Lab Preparations
ISOLATION OF DNA
(Components E-H)
If a precipitate has formed in the DNA extraction buffer,
warm at 37C to redissolve.
1. Add 200 l of DNA Extraction Buffer (H) to each
tube of Proteinase K (F) and allow the pellets to
hydrate for a couple of minutes. Add the dissolved
Proteinase K back to the 10 ml of DNA Extraction
Buffer and mix. Aliquot 1 ml for each group and
keep tubes on ice.
2. Aliquot 1 ml of NaCl solution (G) for each group.
Materials for DNA Isolation
Each student group should receive:
1 ml DNA extraction buffer
1 ml NaCl solution
1 ml TE buffer
2 Microcentrifuge tubes with pestles
4 1.5 ml microcentrifuge tubes with caps
Materials for PCR
Each student group
should receive:
50 l GMO Primer Mix
20 l Gel load solution
2 PCR beads
35 l 100 bp ladder
POLYMERASE CHAIN REACTION
(Components A-D)
Aliquot reagents for PCR.
1. GMO Primer Mix (B)
Aliquot 50 l for each group
For the group performing the control reaction, aliquot an addi-
tional 20 l.
2. Aliquot 20 l gel load solution for each group
3. Aliquot 35 l of 100 bp ladder (C) for each student group.
4. Thaw the positive control sample (D) and dispense 7 l for the one
group performing the control. Place on ice.
Notes and Reminders:
Accurate temperatures and cycle times are critical. A pre-run for one cycle (approx. 3 to 5
min) is recommended to check that the thermal cycler is properly programmed.
For thermal cyclers which do not have a top heating plate, it is necessary to place a layer of wax
above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix
entitled "Preparation and Handling PCR Samples with Wax ".
3. Aliquot 1 ml of TE buffer (E) for each group
4. Place bottles of 95% and 70% Isopropyl alcohol on ice or in the freezer.
Chill thoroughly.
There is enough material for ten student groups sharing ve gels (two groups
per gel). There is material to perform one positive control DNA reaction for PCR.
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Experiment Results and Analysis
* Note:
Depending on the PCR conditions used, a diffuse, small-molecular weight band, known
as a "primer dimer", may be present below the 100 bp marker (shown in the idealized
schematic at the bottom of the gel). This is a PCR artifact and can be ignored. Other
minor bands may also appear due to nonspecic primer binding and the subsequent
amplication of these sequences.
Lane 1 100 bp ladder
Lane 2 GMO marker
(500 bp plant chloroplast, 200 bp CMV 35S
and 127 bp NOS terminator)
Lane 3 GMO food (corn akes)
(Positive for plant chloroplast, CMV 35S
and NOS terminator)
Lane 4 non-GMO food
(Positive for plant chloroplast only)
Lane 5 GMO food (soy chips)
(positive for plant chloroplast, CMV 35S
and NOS Terminator)
500 bp
100 bp
1 2 4 3 5 6
In this experiment, results will vary depending upon the type of genetic modi-
cation (if any) in the food source chosen by the student(s). Successful genomic
DNA purication from foodstuffs can have a signicant impact on the PCR am-
plication and gel electrophoresis results. Poor results and quality of extracted
genomic DNA can be caused by an unsuccessful extraction attempt. For optimal
DNA preparation, particular attention should be paid to the extraction process as
described in the protocol.
*
Please refer to the kit
insert for the Answers to
Study Questions
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Experiment
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Notes
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Experiment #
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Identication of Foodstuffs
from Genetically Modied Organisms
Appendices
A PCR Success Guidelines
B Polymerase Chain Reaction Using Three Waterbaths
C Preparation and Handling of PCR Samples With Wax
D 2.0% Agarose Gel Electrophoresis Reference Tables
E Buffer and Agarose Quantity Preparations
F Agarose Gel Preparation
G Staining and Visualization of DNA with InstaStain Ethidium
Bromide Cards
H Staining and Visualization of DNA - FlashBlue Liquid Stain
I InstaStain Blue: One Step Staining and Destaining
J Electrophoresis Hints and Help
Material Safety Data Sheets
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Experiment
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Appendix
EDVOTEK experiments which involve the amplication of DNA are extremely relevant,
exciting and stimulating classroom laboratory activities. These experiments have been per-
formed successfully in many classrooms across the country, but do require careful execution
because of the small volumes used. The following guidelines offer some important sugges-
tions, reminders and hints for maximizing success.
THE PCR REACTION
1. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward
and reverse primers) and the cell DNA (supernatant) as specied in the experimental
procedures to the microcentrifuge tube containing the PCR reaction bead. Make sure
that the bead (which contains the Taq DNA polymerase, the 4XdTPs, Mg and the PCR
reaction buffer) is completely dissolved. Do a quick spin in a microcentrifuge to bring
the entire sample to the bottom of the tube. Prepare the control reaction similarly.
2. The Thermal cycler: It is critical that the thermal cycler be accurately programmed for
the correct cycle sequence, temperatures and the time for each of the cycles.
3. Oil or Wax: For thermal cyclers that do not have a top heating plate, the reaction in
the tubes must be overlaid with oil or wax to prevent evaporation.
4. Manual Water Bath PCR: Three water baths can be used as an alternative to a ther-
mal cycler for PCR, but results are more variable. Samples require oil or wax layers.
This method requires extra care and patience.
GEL PREPARATION AND STAINING
5. Concentrated agarose: Gels of higher concentration (> 0.8%) require special atten-
tion when dissolving or re-melting. Make sure that the solution is completely clear of
clumps or glassy granules. Distorted electrophoresis DNA band patterns will result
if the gel is not properly prepared.
6. Electrophoretic separation: The tracking dye should travel at least 4 cm from the
wells for adequate separation before staining.
7. Staining: Staining of higher concentration gels (> 0.8%) require special care to
obtain clear, visible results. After staining (15 to 30 min.) with InstaStain Ethidium
Bromide, examine the results using a UV (300 nm) transilluminator. Repeat the stain-
ing as required.
8. DNA ladders or markers: After staining the agarose gel, the DNA 100 bp ladder, 200
bp ladder, or Standard DNA Markers should be visible. If bands are visible in the lad-
der or marker and control lanes, but bands in the sample lanes are faint or absent,
it is possible that DNA was not successfully extracted from the cells. If the ladder
or marker, control and DNA bands are all faint or absent, potential problems could
include improper gel preparation, absence of buffer in the gel, improper gel staining
or a dysfunctional electrophoresis unit or power source.
PCR Experimental Success Guidelines
A
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Experiment
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Appendix
B
PREPARATION OF THE PCR REACTION:
1. The PCR reaction sample should be prepared as specied in the experiment in-
structions. Each PCR reaction sample contains three critical components:
PCR Reaction pellet
Primer mix
DNA for amplication
2. After adding the components of the PCR reaction sample, use clean forceps to
transfer one wax bead to the PCR tube. At the start of the PCR reaction, the wax
will melt and overlay the samples to prevent evaporation during heating.
POLYMERASE CHAIN REACTION CYCLING
3. In the three-waterbath PCR method, the PCR reaction sample is sequentially
cycled between three separate waterbaths, each set at different temperatures, for
a specied period of time. The sequential placement of the reaction sample in
the waterbaths maintained at three different temperatures constitutes one PCR
cycle. One example of a PCR cycle might be as follows:
94C for 1 minute
50C for 1 minute
72C for 1 minute
See experiment instructions for specic program requirements.
4. The PCR tube must be handled carefully when sequentially cycled between the
three waterbaths. For each cycle:
Carefully place the PCR tube in a waterbath oat. Make sure that the sample
volume is at the bottom of the tube and remains undisturbed. If necessary,
pulse spin the tube in a balanced microcentrifuge, or shake the tube to get all
of the sample to the bottom of the tube.
Use forceps to carefully lower the waterbath oat (with tubes) sequentially
into the waterbaths.
5. Process the PCR reaction sample for the total number of cycles specied in the ex-
periment instructions. On the nal cycle the 72C incubation can be extended
to 5 minutes.
6. After all the cycles are completed, the PCR sample is prepared for electrophoresis.
Polymerase Chain Reaction Using Three Waterbaths
Superior PCR results are obtained using an automated thermal cycler. However, if you do
not have a thermal cycler, this experiment can be adapted to use three waterbaths (Cat.
# 544). Much more care needs to be taken when using the three-waterbath PCR method.
The PCR incubation sample is small and can easily be evaporated. Results using three wa-
terbaths are often variable. Please refer to the Appendix entitled "PCR Samples with Wax
Overlays" for sample handling and preparation tips.
Each PCR Reaction pellet
contains Taq DNA poly-
merase, four deoxytriphos-
phates, Mg
+2
and buffer.
It is imperative that the
temperatures are accurately
maintained throughout the
experiment.
Important Note
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Experiment
Identication of Foodstuffs
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Appendix
C
Preparation and Handling of PCR Samples With Wax
For Thermal Cyclers without Heated Lids, or
PCR Using Three Waterbaths
Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appropri-
ate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small
sample volumes from evaporating. For thermal cyclers without heated lids, or when conducting PCR by the
three-waterbath method, it is necessary to add a wax bead to the reaction sample. During the PCR process, the
wax will melt and overlay the samples to prevent evaporation during heating.
Each PCR Reaction
pellet contains Taq
DNA polymerase, four
deoxytriphosphates,
Mg
+2
and buffer.
PREPARING THE PCR REACTION:
1. The PCR reaction sample should be prepared as specied in the experiment
instructions. Each PCR reaction sample contains the following three critical
components:
PCR Reaction pellet
Primer mix
DNA for amplication
2. After adding the components of the PCR reaction sample, use clean forceps to
transfer one wax bead to the PCR tube.
3. Process the PCR reaction sample for the total number of cycles specied in the
experiment instructions.
PREPARING THE PCR REACTION FOR ELECTROPHPORESIS:
4. After the cycles are completed, transfer the PCR tube to a rack and prepare
the PCR sample for electrophoresis.
Place the PCR tube in a 94C waterbath long enough to melt the wax over-
lay. Use a clean pipet to remove most of the melted wax overlay.
Allow a thin layer of the wax to solidify.
Use a clean pipet tip to gently poke a hole through the solidied wax.
Remove the tip.
Use another clean pipet tip to enter the hole to remove the volume of
mixture specied in the experiment instructions. Transfer this volume to a
clean tube.
Add other reagents according to experiment instructions, if applicable,.
Add 5 l of 10x Gel Loading solution to the sample and store on ice.
5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as
specied in the experiment instructions.
29
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Appendix
Time and Voltage Guidelines
(2.0% Gel)
Time Volts
125
70
50
40 min
2.0 hrs
3.0 hrs
Approx. migration
distance of
tracking dye
Gel Size (cm)
Volume
7 x 7
25 ml
7 x 14
50 ml
125
70
50
60 min
2.5 hrs
3.5 hrs
4.5 cm
6.0 cm
Table
C.3
If preparing a 2.0% gel
with concentrated (50x)
buffer, use Table A.7.
If preparing a 2.0% gel
with diluted (1x) buffer, use
Table A.8.
2.0% Agarose Gel Electrophoresis Reference Tables
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.3 for 2.0%
agarose gels. The time for electrophoresis will
vary from approximately 15 minutes to 2 hours
depending upon various factors. Conduct the
electrophoresis for the length of time determined
by your instructor.
For DNA analysis, the recom-
mended electrophoresis buffer is
Tris-acetate-EDTA, pH 7.8. The
formula for diluting EDVOTEK
(50x) concentrated buffer is one
volume of buffer concentrate to
every 49 volumes of distilled or
deionized water. Prepare buffer
as required for your electropho-
resis unit.
D
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 14
0.5
1.0
0.5
1.0
24.5
49.0
+
= +
Table
A.7
25
50
Individual 2.0%
UltraSpec-Agarose Gel
Table
A.8
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 14
0.5
1.0
25
50
+
Individual 2.0%
UltraSpec-Agarose Gel
50x Conc.
Buffer (ml)
Distilled
Water (ml)
+
EDVOTEK
Model #
Total Volume
Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36
Dilution
Table
B
300
400
1000
294
392
980
6
8
20
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Experiment
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Appendix
E
To save time, electrophoresis buffer and agarose gel solution can be prepared in larger
quantities for sharing by the class. Unused diluted buffer can be stored for use at a later
time and solidied agarose can be remelted.
Buffer and Agarose Quantity Preparations
BULK ELECTROPHORESIS BUFFER
Quantity (bulk) preparation for 3 liters of 1x electropho-
resis buffer is outlined in Table D.
Concentrated
Buffer (50x)
(ml)
Distilled
Water
(ml)
Total
Volume
(ml)
60 2,940 3000 (3 L)
=
+
Bulk Preparation of
Electrophoresis Buffer
Table
D
60C
8.0 8.0 392 400
Batch Preparation of
2.0% UltraSpec-Agarose
Amt of
Agarose
(g)
Concentrated
Buffer (50X)
(ml)
+
Distilled
Water
(ml)
Total
Volume
(ml)
= +
Table
E.3
PREPARING AGAROSE GELS BY BATCH
For quantity (batch) preparation of agarose gel solu-
tion, refer to Table E.3.
1. Use a 500 ml Pyrex ask or beaker to prepare the
diluted gel buffer
2. Pour the appropriate amount of UltraSpec-Agarose into the prepared buffer. Swirl to disperse
clumps.
3. With a marking pen, indicate the level of solution volume on the outside of the ask.
4. Heat the agarose solution in the same manner as described for individual gel preparation. The heating
time will require adjustment due to the larger total volume of gel buffer solution.
5. Cool the agarose solution to 60C with swirling to promote even dissipation of heat. If
evaporation has occurred, add distilled water to bring the solution up to the original
volume as marked on the ask in step 3.
6. Dispense the required volume of cooled agarose solution for casting each gel. The
volume required is dependent upon the size of the gel bed.
7. Allow the gel to completely solidify. It will become rm and cool to the touch after
approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.
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Experiment
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Appendix
F
EDVOTEK electrophoresis units
include 7 x 7 cm or 7 x 14 cm
gel casting trays.
A. Using Rubber dams:
Place a rubber dam on each end of the bed. Make sure the rubber dam ts
rmly in contact with the sides and bottom of the bed.
B. Taping with labeling or masking tape:
Extend 3/4 inch wide tape over the sides and bottom edge of the bed.
Fold the extended tape edges back onto the sides and bottom. Press contact
points rmly to form a good seal.
If gel trays and rubber end
caps are new, they may be
somewhat
difcult to
assemble.
Here is
a helpful
hint:
2. Place a well-former template (comb)
in the rst set of notches at the end
of the bed. Make sure the comb sits
rmly and evenly across the bed.
Preparing the Gel bed
1. Close off the open ends of a clean and dry gel bed (casting tray) by
using rubber dams or tape.
Agarose Gel Preparation
Step by Step Guidelines
Place one of
the black end
caps with the
wide u shaped slot facing up on
the lab bench.
Push one of the corners of the gel
tray into one of the ends of the
black cap. Press down on the tray
at an angle, working from one end
to the other until the end of the
tray completely ts into the black
cap. Repeat the process with the
other end of the gel tray and the
other black end cap.
Casting Agarose Gels
3. Use a 250 ml ask or beaker to prepare the gel solution.
4. Refer to the appropriate Reference Table (i.e. 0.8%, 1.0% or 2.0%)
for agarose gel preparation. Add the specied amount of agarose
powder and buffer. Swirl the mixture to disperse clumps of agarose
powder.
5. With a lab marking pen, indicate the level of the solution volume on
the outside of the ask.
6. Heat the mixture to dissolve the agarose powder.
A. Microwave method:
Cover the ask with plastic wrap to minimize evaporation.
Heat the mixture on High for 1 minute.
Swirl the mixture and heat on High in bursts of 25 seconds
until all the agarose is completely dissolved.
B. Hot plate method:
Cover the ask with aluminum foil to minimize evaporation.
Heat the mixture to boiling over a burner with occasional
swirling. Boil until all the agarose is completely dissolved.
Continue heating until the nal solution appears clear (like water) with-
out any undissolved particles. Check the solution carefully. If you see
"crystal" particles, the agarose is not completely dissolved.
At high altitudes, it is recommended
to use a microwave oven to reach
boiling temperatures.
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Experiment
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Appendix
F
7. Cool the agarose solution to 60C with careful swirling to
promote even dissipation of heat. If detectable evaporation
has occurred, add distilled water to bring the solution up to the
original volume marked in step 5.
After the gel is cooled to 60C:
If you are using rubber dams, go to step 9.
If you are using tape, continue with step 8.
DO NOT
POUR
BOILING
HOT
AGAROSE
INTO THE
GEL BED.
Hot agarose solution
may irreversibly warp
the bed.
60C
+
Black Red
Sample wells
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39
962
Experiment
Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
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Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
962
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