Module 4 Manipulation and Analysis of DNA

Goals for Module 4

1. To review the basic structure of deoxyribonucleic acids (DNA) and the organization of
genetic material within cells.
2. To understand what a plasmid is and the central role of plasmids in mediating antibiotic
resistance.
3. To engage in a simple cloning experiment including the isolation and manipulation of
plasmid DNA.
4. To determine the concentration of DNA by spectroscopy using a Nanodrop
spectrophotometer
5. To perform restriction digestion and analysis of plasmid DNA by agarose gel
electrophoresis
6. To conduct PCR reactions in order to amplify a target segment of DNA
7. To perform ligations of DNA
8. To transform bacteria by introducing plasmids so that the bacteria express new proteins.

Introduction and Background

Relevance
The ability to isolate small segments of DNA from a complex mammalian genome has become
an essential tool for analyzing the molecular basis of cancer, for identifying mutations that
underlie hereditary forms of disease, and for exploring gene structure-function relationships.
DNA cloning techniques are used to investigate the intricate regulation of gene expression
during normal development as well as during pathogenic processes.

These types of studies are possible because of:

1. The identification and commercial production of enzymes that cut (restrict) DNA at
highly specific sites,
2. The ability to insert the DNA fragments that are produced into small non-chromosomal
replicating DNA molecules found in bacteria (plasmids). The plasmid functions as a
carrier to replicate the inserted segment of DNA,
3. The ability to amplify miniscule amounts of DNA starting material into much larger and
more useful quantities.

Review of DNA structure and organization:

As part of this module, we would like you to understand the significant differences in the
genomes of bacteria vs higher eukaryotes and the technical skills required to work with their
DNA. DNA is a linear polymer composed of purine (adenine and guanine) and pyrimidine
(thymidine and cytosine) bases attached to deoxyribose sugars. These units are termed
nucleotides and are covalently linked by phosphodiester bonds (figure 1top). The DNA strand is

1
stabilized by hydrogen bonding with a complementary DNA strand to form a double stranded
helix.

The linear sequence of nucleotides contains the information specifying the sequence of amino
acids that form proteins. Three nucleotides are used to code for each amino acid and this is
know as the triplet code. The segments of protein-coding DNA are called genes.

Local unwinding of the two helical strands exposes single strands of DNA which can be used as
templates to replicate the original double stranded DNA molecule (figure 1 bottom).

Figure One. Structure of DNA

adapted from: Recombinant DNA A short course; JD Watson, J Tooze, DT Kurtz, &
Molecular Cell Biology; 5th edition H Lodish et al

In bacteria the complete genome is usually a single circular piece of double stranded DNA. This
bacterial chromosome is concentrated by folding into a small region of the cytosol termed the
nucleoid. Bacteria sometimes carry small non-chromosomal replicating circular DNA molecules
called plasmids which the bacteria use, among other things, for conferring antibiotic resistance.
These plasmids have become an important tool of modern molecular biology because of their
ease of isolation and manipulation.

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In humans and other eukaryotes, the organization of the genome is much more complex. For
eukaryotic organisms, the genome is partitioned into segments of DNA that are packaged into
highly ordered structures termed chromosomes. Chromosomes are confined in a discrete
membrane bound organelle, the nucleus. Because of the length of DNA and complexity of gene
structure, the chromosomal DNA is organized and packed into repeating, protein-associated
subunits. The protein:DNA subunits are further compacted and assembled in regional loop
domains that are attached along protein scaffolds (Figure 2 left). The central scaffold resembles
an internal skeleton for the chromosome (Figure 2 right). This organized and tightly assembled
protein-DNA structure is called chromatin. Simply accessing the chromosomal regions for gene
expression while actively sequestering other regions requires extensive regulation. Thus,
chromosome structure is dynamic, with regions opening for transcriptional activity and
expanding or condensing during cellular replication.
These prokaryote-vs-eukaryotic differences in size, organization, and complexity of the genome
require different techniques to isolate and manipulate DNA.

Figure Two. Model for packing of chromatin and the chromosome scaffold in
eukaryotic metaphase chromosomes

adapted from: Recombinant DNA A short course; JD Watson, J Tooze, DT Kurtz, &
Molecular Cell Biology; 5th edition H Lodish et al

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Introduction to recombinant DNA techniques
By cloning DNA scientists can study the structure, function, and regulation of selected
mammalian gene segments in isolation from the highly complex mammalian chromosome.

In addition to their own chromosome, bacteria can carry tiny circular DNA molecules that
encode relatively few genes and are only several thousand base pairs in total size. These
molecules are small replicating units called plasmids. Plasmids were first identified by their
ability to confer antibiotic resistance. Most plasmids replicate at high numbers in bacteria and
produce sufficient inhibitory enzyme to achieve antibiotic resistance. Because of their small size,
simplicity, and large numbers, bacterial plasmids are a useful vector to replicate small segments
of DNA in amounts large enough for physical isolation and characterization (figure 3).

Figure 3. Structure of a bacterial plasmid

adapted from: Recombinant DNA A short course; JD Watson, J Tooze, DT Kurtz, & Molecular Cell Biology; 5th
edition H Lodish et al

Enzymes to cut and modify the ends of DNA for cloning

Restriction enzymes recognize specific short DNA sequences and cleave the phosphodiester
bonds at that site. Restriction enzymes can be used to digest long DNA molecules into smaller
fragments that can be manipulated for analysis. A general cloning strategy employs plasmids that
have been engineered with a set of unique sites that are cut by different restriction enzymes.
Foreign DNA fragments can be digested with the same restriction enzyme in order to have
complementary sites with the plasmid so that they can fit together. The general steps are
outlined in the top of figure 4.

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 Figure 4. Transfection and selection of bacteria containing plasmids

adapted from: Recombinant DNA A short course; JD Watson, J Tooze, DT Kurtz, & Molecular Cell Biology; 5th
edition H Lodish et al

There is a vast collection of commercially available restriction enzymes that digest DNA at
different recognition sequences and provide a variety of specific ways to digest a target DNA for
cloning. We will use these techniques to analyze a plasmid that carries a gene encoding green
fluorescent protein (GFP) that is used extensively in research. The experiments will use two
restriction enzymes- Nco I and Not I.

Ligation, the molecular glue

Plasmid DNA and the larger target DNA that are digested with the same restriction enzyme have
compatible ends. These two digested DNAs can be linked by an enzyme called DNA ligase. The
normal cell function of DNA ligase is to link segments of replicating genomic DNA or DNA that

5
must undergo some form of repair. When used with plasmid DNA, ligation reactions potentially
generate an intact recombinant plasmid molecule that now carries a new piece of genomic DNA.

An essential step in cloning is to be able to re-form the phosphodiester bonds between the
restriction digested vector and fragments of DNA that are to be inserted into the vector. Highly
concentrated DNA ligase is available that we will use to reform the phosphodiester bond
between the plasmid cut with restriction enzymes and the digested insert DNA sequence (top two
steps in figure 4).

Transformation- the internalization of DNA

Transformation is a relatively simple procedure to introduce plasmids back into carrier bacteria
for growth in order to increase the amount of plasmid DNA for further analysis.
Bacteria can take up exogenous plasmids when the bacteria are grown at a low density and then
incubated in combinations of CaCl2 and/or MgCL2 or LiCl2. It is thought that the divalent ions
swell and semi-permeabilize the bacterial walls allowing influx of the plasmid DNA. The CaCl2
altered cell wall can recover its structural integrity after the divalent salts are diluted out by
placing the cells into a nutrient culture media.

The Quest - finding your clone by selection and screening

Not all the bacteria will necessarily be successfully transfected and even those that are may be
transfected with plasmids that are not functional in the way they were designed to be. To deal
with these possibilities, cloning new DNA into a plasmid that encodes antibiotic resistance (R)
offers a great advantage for detection of just those bacteria with the desired plasmid.
Transformed bacteria can be spread to grow on nutrient petri plates with the selective antibiotic
in the media. The antibiotic sensitive bacteria will die and the subset of bacteria that now harbor
the R-plasmid will not. Only these antibiotic resistant bacteria will form colonies (step 3 figure
4). These survivor colonies can be further screened to identify R plasmids that now carry the
inserted genomic DNA.

Plasmid isolation - selective disruption followed by either alcohol precipitation or affinity

binding
The first step in purifying an R plasmid entails picking individual colonies and putting them into
broth cultures for growth overnight. The next day the bacteria are pelleted out of the media and
re-suspended in a buffer. A lysis solution (soap & buffer) is added that partially breaks up the
cell structure and cell components. The ruptured cell debris can be precipitated out of solution
under conditions where small molecules such as the plasmids remain in solution for recovery.
The recovery of plasmids can be accomplished in either of two ways:

1. By binding the DNA to an affinity column, washing unwanted material off of the column
and then eluting the bound plasmid off of the column matrix
2. or by combining high salt and ethanol to precipitate the plasmid DNA out of solution.

We will use a small scale affinity column with plasmid binding and elution.

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Detection of plasmid DNA by gel electrophoresis
How do you determine your success at isolating plasmid DNA and how do you determine the
purity of the plasmid prep? As an initial screen, you can run the intact plasmid on an agarose gel.
In this module we will provide a separate guide on steps for making and running an agarose gel.
The larger a plasmid is, the slower it will migrate through the agarose gel.

As you know from the polyacrylamide gels you ran in module 3, electrophoresis is the separation
of molecules based upon their migration through a matrix in an electrical field. Agarose is a
polysaccharide polymer that is a refined extract of agar, similar to what was used to form the
bacterial growth media in module 2. Agarose can be re-suspended in a buffer, heated to boiling
and cooled, at which point it polymerizes into a gel. This gel slab functions as a molecular sieve
to separate different sized DNA fragments subject to an electric current. The DNA migrates
through the pores of the gel matrix based upon both the size and charge. Different sized DNA
molecules can be seen as bands in the gel when stained with a DNA binding dye and exposed to
UV light. By using DNA markers of known size you can estimate the apparent size or carefully
measure, graph, and calculate the size of DNA bands (figure 5).

Figure 5. Sizing DNA on an Agarose Gel

adapted from: Recombinant DNA A short course; JD Watson, J Tooze, DT Kurtz, &
Molecular Cell Biology; 5th edition H Lodish et al

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Polymerase chain reaction (PCR), making a mountain out of a molehill
A rapid technique to generate useable amounts of DNA is to use the polymerase chain reaction
(see figure 6 below). PCR uses a process with the enzyme DNA polymerase to repetitively copy
a DNA sequence from extremely small amounts of starting material. Small synthetic single
stranded DNA primers that are complementary to the target DNA (in this case our GFP gene) are
used to direct DNA synthesis. Therefore, this technique requires that the investigator already
have some DNA sequence information in order to choose and synthesize DNA primers for the
polymerase enzyme.

During a cycle of heating/cooling the short DNA primers anneal (i.e. bind) to a strand of DNA
that will be copied. Then a thermostable DNA polymerase extends the DNA sequence from the
specific primers after which the new DNA strand is separated from the old one by heating.
Repeated cycles of annealing, synthesis, and dissociation eventually generate useful amounts of
the DNA fragment of interest from just pico/femto gram amounts of starting template.

PCR can amplify DNA from a complex genome that was present in only vanishingly small
amounts. PCR is used to rapidly screen clones (or other DNA samples) to identify the desired
clone by gel electrophoresis.

In this laboratory module you will manipulate a commonly used reporter gene: enhanced green
fluorescent protein (EGFP). EGFP is a bio-fluorescent protein that was originally isolated from
jelly fish. This protein has widespread utility in genetics as a read-out of gene expression.
Expression of EGFP protein is achieved by introducing a plasmid into cultured cells or tissue
where a mammalian promoter drives EGFP transcription.

8
 Figure 6. The polymerase Chain Reaction

adapted from: Recombinant DNA A Short course; JD Watson, J Tooze, DT Kurtz, & Molecular Cell Biology; 5th
edition H Lodish et al

9
 Lab Activities (days 1 & 2)
Overview
The first two days of lab activities in Module #4 are broken down into four parts:
1. Recovery and analysis of plasmid DNA
a. Plasmid isolation
b. DNA isolation
2. Agarose gel electrophoresis of intact plasmids to detect plasmid yield & determine size
3. Bacterial Transformation
4. Quantitation of DNA using Nanodrop

Part 1: Recovery and analysis of plasmid DNA

This two-day laboratory session is the first of several that will familiarize you with the basic
techniques of manipulating and cloning DNA. Today you will extract plasmid DNA from
bacteria. You will learn how to conduct agarose gel electrophoresis and how to get bacteria to
internalize plasmids (transformation). In subsequent laboratory exercises you will restriction
digest and map DNA, PCR amplify a gene encoding a green fluorescent protein (EGFP), and
ligate DNA molecules.

As starting material, you will be given two different cultures of bacteria harboring plasmids that
were grown in an antibiotic selective media. One plasmid is pEGFP-N1. This plasmid carries
the EGFP gene cassette that we will PCR amplify and save to use in Module #5 on cell biology
where it will be used for gene expression studies in mammalian cells. A second plasmid is
named pUC19 that is a high copy-number plasmid which serves as a positive control for
isolation and recovery of plasmid DNA.

Plasmid Isolation from bacteria using affinity column chromatography

There are several strategies to reliably isolate plasmid DNA from bacteria. One common
approach (which we will not be using here) involves the rapid lysis of bacteria and selective
removal of cellular components by salt precipitation of large molecular weight molecules. The
precipitation step is followed by recovery of the relatively smaller plasmid DNA molecules from
the supernatant by EtOH precipitation.

We will use a more efficient modified alkaline lysis that includes adsorption of DNA in high salt
conditions onto a premade silica matrix spin column. Once the plasmid has been bound to the
column in high salt, adding a buffer without salt results in release & elution of the plasmid DNA
from the column matrix. This results in recovery of the plasmid in a small volume and avoids
the EtOH precipitation step. You will use a small-scale commercial kit from New England
Biolabs.

At the start of the lab the instructors will provide a brief demo on operating the table top
centrifuges.

DNA isolation on the spin column

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Preparation prior to starting:
Add 4 volumes of EtOH to one volume of the plasmid wash buffer 2 (e.g. add 20ml 95% EtOH
per 5ml buffer). This will have been done in advance for you by an instructor.

You will be working with 2 different plasmids simultaneously. Therefore, it is important that
you label all tubes as you proceed: pEGFP, and pUC19. When it comes time to centrifuge your
samples, you can use the two plasmid samples to balance each other in the centrifugation steps.

1. Pellet 1.5 ml of overnight culture in the table top micro-centrifuge at 6,000 rpm for 2
minutes.

2. Pipet off the media and add 200 ul of plasmid resuspension buffer B1.
Gently pipet the bacterial pellet up and down until there are no clumps of bacteria.

3. Add 200 ul of plasmid lysis buffer B2 and mix by rapid inversion until the turbid solution
just becomes a clear pink color, this typically requires 6-8 inversions in less than 1
minute. Mix but do not vortex!

4. Add 400 ul of plasmid neutralization buffer B3 and again mix by rapid inversion. You
will see the solution turn yellow and a white precipitate form.

5. Centrifuge for 3 minutes at 6,000 rpm in the table top micro-centrifuge. Always balance
the tubes in the centrifuge.

6. While the cell lysates are centrifuging prepare two plasmid affinity mini-columns by
placing the columns in a 1.5 ml centrifuge tube, one for each plasmid. Label each affinity
column.

7. When the centrifugation of cell lysates is completed, remove the tubes from the
microfuge. Slowly pipet 5-600ul out of the tube without disturbing the pelleted material.
Transfer this cleared supernatant into the spin column.

8. Centrifuge the spin column for 60 seconds (5-6,000 rpm) and discard the flow-through,
then re-insert the column into the carrier tube.

9. 9.Wash the spin column with 200 ul of the plasmid wash buffer 1, centrifuge for 60
seconds (6,000 rpm) and discard the flow through. Add 400 ul of wash buffer 2 and
centrifuge for 60 seconds.

10. Transfer the spin column to a clean/new centrifuge tube. Recover the plasmid DNA
from the spin column by adding 50 ul of DNA elution buffer into the column. Allow the
column to stand for at least 1 minute and then centrifuge for just 60 seconds.

The 50ul of buffer containing eluted plasmid DNA will now be in the bottom of the tube, ready
for analysis. Remove the Monarch spin column/cartridge. Cap and Close the tube containing
your DNA and make sure that each tube is clearly labeled.

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In later laboratory sessions, the DNA prep will be evaluated by UV spectroscopy with the
Nanodrop spectrophotometer and then the DNA will be digested with restriction enzymes & used
for PCR amplification.

Part 2: Agarose gel electrophoresis of intact plasmids

The goal in this part is to determine the plasmid yield from part 1 and to determine the plasmid
size.
You will use a Tris-Acetate, EDTA buffer (TAE), pH 7.9 as the electrophoresis buffer. You will
need approximately 300 ml of buffer for the electrophoresis chamber. Dilute from our stock 50X
TAE concentrate if the buffer has not already been pre-diluted.

A diagram of the steps involved in pouring a gel, loading samples and running the gel is shown
in figure 7.
Figure 7

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Adapted from: The Nature of Biology- Biology, J. Byette, University of Cincinnati Department of Biological
Sciences
Preparation of a 1% agarose gel.
The agarose in TAE buffer will be prepared ahead of time and stored in a 550 incubator/oven.
Pour out 40 ml into a disposable 50 ml screw-cap tube. Add 5 ul of the stock ethidium bromide
(EtBr) to the tube, cap, and invert to mix. EtBr binds to the DNA for visualization under UV
light.
An instructor will provide the EtBr.

Warning: EtBr is a DNA intercalating agent and potential mutagen. Always wear gloves.
We have special disposal bags and containers for biohazard disposal of the gel after the
electrophoresis run is completed and photographed.

The instructor will be available to help with the setup of the gel tray and insertion of combs that
form the sample wells in the gel slab. The gel will take 15+ minutes to polymerize and will turn
translucent. Ask a TA to assist with deciding when the gel has fully solidified. At that point it is
ok to gently remove the comb from the gel.

Loading your samples onto the gel

For each of your two plasmid preparations, prepare a sample to load onto the gel by mixing the
following reagents:
1ul of the plasmid prep
3 ul of buffer TE
1 ul of the gel loading dye. (The dye is a dense dark blue marker that allows the DNA
sample to sink through the buffer into the wells and allows you to track the migration of
the samples through the gel.

For reasons of pipetting accuracy, you may use larger volumes than those above, but only 5 ul of
the mixture will be loaded into each well.

You will also be given a sample of cccDNA size markers for loading onto the gel. These
markers will allow you to determine the size and relative yield of your plasmids.

Load 5 ul of each of your two plasmid samples into wells on the agarose gel and on either side of
the plasmid wells load 5 ul of the cccDNA sample. Suggestion: The sample wells are quite
narrow and there is not much space to fill the well without stabbing through the bottom of the
well or pipetting over the well where the sample is lost into the buffer and washes away. Practice
gel loading with TE and blue loading dye alone if there is time.

Running the gel

1. Place the lid onto the loaded gel, plug leads into the power pack and turn voltage to 50 V.
2. When the dye front has migrated approximately 1/2 the length of the gel, stop the run by
turning off the power pack
3. Move the gel to a transport tray so that the gel does not slip out of the tray.

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 4. Observe the DNA under UV light. (An instructor will help you make sure that the UV
blocking cover is in place.)

Use your cell phone to photograph through the UV resistant cover.

Based upon the ladder of cccDNA plasmid markers estimate the size of your plasmids and
check with the instructor to determine if your results are similar to the known (and expected)
sizes. Use a mm ruler to measure the distances that the plasmids migrated from the origin so that
you can graph and more precisely determine the sizes of the plasmids for your lab report.

Part 3: Bacterial transformation, how to incorporate plasmids into bacteria

Bacterial transformation refers to the process of inducing live bacteria to internalized plasmids.
The intrinsic ability of bacteria to take up and replicate foreign DNA is modest. However,
bacteria can be made competent to take up plasmids. This involves variations on the theme of
growing the bacteria to low density followed by incubating them in CaCl2 or LiCl2 solutions. For
this laboratory you will use commercially available competent cells. The cells are fragile and
sensitive to any warming condition. Keep the competent cells on ice at all times.

Key to detecting and tracking the presence of a plasmid is for the plasmid to encode a selectable
marker that confers a growth property not present in the stock bacteria. This is almost always a
gene encoding antibiotic resistance. Thus, spreading bacteria on a petri plate containing the
antibiotic kills all of the bacteria except for those that have internalized plasmid DNA and are
expressing antibiotic resistance. The plasmid that we will use in this experiment encodes
resistance to ampicillin. The addition of 50-100ug/ml of ampicillin to the LB media petri plates
permits the selection-growth of colonies that have taken up the plasmid and express the Amp
resistance gene.

The transfection protocol

1. Remove cells from storage in the freezer and put on ice at time of use.

2. Label two sterile 14ml plastic snap-cap tubes (+DNA, -DNA) and place on ice. Be sure
to chill tubes before adding cells.

3. Pipet 25 ul of the competent cells into each tube. Return the vial of cells to the freezer for
other lab teams to use.

4. To the + DNA tube add 1 ul of plasmid DNA (pUC19), to the no DNA tube add 1ul of
TE buffer and hold on ice 30 minutes.
5. The DNA is diluted in TE (10mM Tris-HCl, 0.1mM EDTA, pH 7.9) and the
concentration of the stock plasmid is 10 pg/ul. To some degree the longer the incubation
period the more uptake/ transforming colonies you will obtain. You can discuss the
predicted number of colonies with the instructor.

6. For a negative control prepare a tube with the competent cells but no plasmid DNA.

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 7. The tubes are moved to a 370 water bath for 30 seconds and then returned to ice for five
minutes.

8. Add 500 ul of bacterial recovery & growth media (SOC media). IF time permits incubate
the bacteria in SOC for 30 minutes prior to plating.

9. Pipet 3 different volumes of the cells+plasmid DNA (50, 100, or 300 ul ? You choose)
onto the surface of 3 ampicillin (Amp) petri dishes and spread the cells using the
technique you learned in module 2. Repeat the spreading of 300ul of cells from the no-
DNA tube on a 4th Amp plate.

Record the amount/volumes and amounts of plasmid DNA used so that you can calculate the
transformation efficiency in the following lab.
After the liquid is absorbed into the surface move the dishes to an incubator set to 370 overnight.

Part 4. For the Following Lab:

1. Complete any work from the experiments above.

2. After overnight incubation the instructor will remove the selection plates to 40 storage
until the follow-up lab. Remove your plates to room temperature.

3. Most commercial cells can produce 1 X 106 or greater transformants per ug of intact ccc
DNA. In this initial experiment you set up transformations using 10 picogram (pg) of
plasmid and titrated down. Count the number of colonies present and calculate the
number of transformants obtained per pg of plasmid DNA used in the transformation and
plating. Express as Amp resistant colonies obtained per ug of plasmid DNA. Include this
result and calculation in your lab report.

cfu / plate X 1x 106pg X transformation vol. X dilution factor = cfu / ug

plasmid
pg pUC19 DNA ug volume plated

Quantitation of Your Plasmid DNA using Nanodrop Technology

You will quantify the concentration and purity of the plasmids. Conventional UV
spectrophotometers required substantial amounts of plasmid sample in the larger cuvettes. The
DNA would then have to be recovered by EtOH precipitation and added extra steps to the
endpoint measurement. The Nanodrop spectrophotometer uses only 1 ul of sample per
measurement and preserves the bulk of material for experiments.

The Nanodrop spectrophotometer consists of a clamshell type cover over a pedestal where the
sample is placed for reading. To load a sample, lift the arm, pipette the sample on the small silver

15
circle and close the clamshell arm back down. Make sure the liquid contacts both the top and
bottom pedestals.

Nanodrop protocol:
1. Open Nanodrop Software from the desktop
2. Click on Nucleic Acid
3. Wipe the upper / lower sample surface (pedestals) with a Kimwipe and load a 2 ul sample
of water to initialize the machine.
4. Click OK and the Nanodrop will function
5. Lift the arm and wipe off the water from the two sample surfaces with a Kimwipe.
6. Load 1-2 ul of the blank (TE buffer or the Qiagen EB buffer) close the arm and click
Blank in the upper left hand corner of the window, the Nano drop will click again.
7. Wipe off the blank fluid from the upper / lower surfaces with a Kimwipe.

DNA Concentration Determination:

8. Load your plasmid DNA sample and click Measure in the upper left hand corner of the
screen. The Nanodrop will click as it measures. The A260, A280, and A260/A280
reading and calculated concentration will be displayed in the lower right hand corner.
Some of our Nanodrops have a printer attachment for recording the results.
9. Wipe the sample off of the surfaces repeat the process for the other plasmid preparations;
the blanks do not need to be repeated.

10. An A260 of 1.0 for dsDNA = 50 ug/ml. Based upon the OD readings and volume
recovered in ul calculate the concentration of your plasmids as ug/ml. Include the
Nanodrop scans and concentration calculation of the plasmids in your lab report.

You can repeat the plasmid isolation or gel if you are not satisfied with your 1st attempt.

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 Lab Activities (days 3 & 4)
Overview
The third and fourth days of lab activities in this Module are broken down into three parts:
1. Digestion of a plasmid using restriction endonuclease enzymes
2. PCR amplification of a gene coding for a green fluorescent protein
3. Analysis of the amplified gene

Goal
The goal of this section of Module 4 is to provide an introduction to the mapping and analysis of
the location of restriction (R) sites along a linear or circular segment of DNA. The relative
position of R sites can be used to select a set of restriction enzymes to digest DNA and generate
fragments for cloning. The restriction map can also guide the selection of isolated DNA
segments to use as molecular probes for in situ hybridization, although we will not be doing that
as part of this lab.

Introduction to Restriction Mapping

Restriction Enzymes. Restriction enzymes are the main way to digest long pieces of DNA at
defined sites for further manipulation. Restriction enzymes cut double stranded DNA at special
types of DNA sequences called palindromes. Palindromes are sequences of things like words or,
in this case nucleotides, which read the same forwards and backwards. The archetypical word
palindrome was written about Napoleon and goes like this: Able was I ere I saw Elba. In the
case of DNA, a palindromic sequence is a nucleotide sequence on double-stranded DNA in
which reading 5' (five-prime) to 3' (three prime) forward on one strand matches the sequence
reading 5' to 3' on the complementary strand. An example of such a nucleotide sequence is
shown in figure 8.
Figure 8

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One way to map sequences of DNA is to select an R site that is unique in a circular plasmid.
Digestion with the restriction enzyme that recognizes that unique site cleaves the circular
plasmid producing a linear molecule with the two ends of the R site at each end (top line of the
figure 9). If the plasmid contains a second restriction enzyme cleavage site, then a mapping
strategy can then be developed in which the plasmid is cleaved with only the first enzyme or
with a combination of the two enzymes followed by gel electrophoresis. A single band is
generated by enzyme 1 cutting and linearizing the circular plasmid. (Figure 9). This band is
altered by digestion with the enzyme 2. R enzyme cut sites are indicated by arrows.

Figure 9

Enzyme 1 (1 band) 1________________________2__________1

Enzymes 1 & 2 = (2 bands) 1________________________2

2__________1

Running the theoretical digests above on a gel would indicate that enzyme 1 cuts once and
linearizes the circular plasmid DNA and that enzyme 2 cuts once approximately the distance
from one end of the enzyme 1 recognition and digestion site.

Repeating this type of experiment with other combinations of restriction enzymes that generate
additional cuts sites allows the investigator to generate a map of the relative position of several R
sites to each other that the investigator can use in future cloning endeavors.

Restriction enzyme reaction dynamics (i.e. Kinetics). How long will you have to run a typical
DNA digestion reaction? This is a function of the amount of DNA, units of enzyme per ul, and
how clean your DNA preparation is.

Most commercially available restriction enzymes have been cloned and purified to be free of
nucleases and other inhibitory material. Restriction enzymes are usually supplied in concentrated
form. The buffer requirements can vary greatly between different enzymes. For each particular
restriction enzyme that one purchases, the reaction condition and buffer requirements have been
optimized for pH, ionic strength, and divalent cation concentrations and the appropriate buffer
(usually at 10X concentration) are supplied.

The R enzymes you will use in this module are called NcoI and NotI and are supplied at
concentrations of 10 units per microliter (U/ul). A unit of activity is defined as the amount of
enzyme that it takes to digest 1ug of DNA in 1 hour at 37 0C. Your agarose gel electrophoresis
conditions should be sensitive enough to detect 50-100 ng of digested DNA. Therefore, one unit
of enzyme should digest 100ng (1/10 of a ug) in about 6 minutes. Since you will be adding
approximately 5 units in each reaction, the digestion should be complete in a very short period of

18
time. The prolonged 30-minute reaction time we are recommending is to assure complete
digestion so that you obtain a clearcut result on your agarose gel run.

Day 3. Plasmid digestion and PCR amplification

Part 1. Protocol for plasmid digestion
From your Nanodrop reading of our starting plasmids you should have had good yields of the
pEGFP-N1 plasmid and you will use the values obtained with this plasmid prep for an initial
mapping experiment.

Approximately 100-150ng of digested DNA is sufficient to be visualized on an agarose gel, so it

is reasonable to set up the digestions using 500+ ug of DNA. For precise measurements you will
need to use the p10 pipetman and the p10 specific tips.

1. ON ICE-Set up the following R digests in 15 ul reactions (final volume). Note: the

same buffer can be used with both the Nco I and NotI enzymes. This buffer is a 10X
concentrate. Therefor each 15 ul reaction volume will need 1.5 ul of the 10X buffer.
2. The example in step 4 below uses 3 ul of plasmid DNA but you can adjust the input
volume of plasmid up or down depending on the Nanodrop data you have obtained for
your sample.

3. The four initial DNA digests are:

pEGFP-N1 with no R enzyme (a control for undigested cccDNA).
pEGFP-N1 with 0.5 ul of the R enzyme Nco I
pEGFP-N1 with 0.5 ul of the R enzyme Not I
pEGFP-N1 with 0.5 ul each of Nco I and Not I

4. For each 15 ul reaction:

Subtract the volume of plasmid, volume of 10x buffer, and volume of enzyme to be
added for each 15 ul reaction. The remainder is the amount of dH20 that you need to add
& bring the reactions to 15ul final volume.

Example:
pEGFP-N1 DNA 3 ul
10X digestion buffer 1.5 ul
dH2O 10 ul
Restriction enzyme 0.5 ul

5. Order of addition:
a. For each reaction add the appropriate amount of dH2O with 10X buffer and plasmid
DNA together
b. Mix briefly by gentle pipetting
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 c. Finally add the restriction enzyme(s) and mix again by pipetting.

6. Insert the tubes in a foam floaters and place in a 370 H2O bath for 20-30 minutes.

7. Prepare and run a 1% agarose gel for electrophoresis of the digested plasmid DNA:
While the plasmid DNA digestion reactions are running, obtain agarose and EtBr, then
pour your agarose gel and allow it to solidify

8. Load the plasmid DNA digests with 2 ul of 1 kB ladder on either side of the pEGFP-N1
digests.

9. Run the gel at 70 V (approx. 35mA). To obtain good resolution of the R enzyme
fragments, slow electrophoresis is best. Allow the gel to run until the blue tracking dye
migrates at least half the length of the gel (2/3 of the gel will give a better resolution of
the digested plasmid bands). Photograph the gel with a mm ruler next to the gel for easy
measurement of migration distances and graphing.

10. An instructor will provide a print reference guide of the various sizes of the DNA bands
in the 1kB ladder to allow you to graph and visually estimate the size of the bands in your
plasmid digestions with NcoI and NotI.

For the lab report

a. Determine how many NotI and Nco I sites are in the plasmid and compare to the 1kB
ladder to estimate the sizes of the fragments.
b. Graph the size of the individual bands relative to the mm of migration of the standards in
the ladder
c. What is the size of the pEGFP-N1 plasmid as determined from the NotI digest?
d. Does the size of the digestion fragments add up to the single cut linearized plasmid?
e. Which Nco I fragment is cut by the R enzyme Not I?

Part 2. Polymerase chain reaction (PCR) amplification of EGFP gene

Goals: In these activities, you will:
a. Prepare PCR reactions that use Taq polymerase to amplify the target gene EGFP. Taq
polymerase is thermo-stabile, meaning that it is not inactivated by high temperatures.
b. Determine the relative amount of template DNA needed to drive a productive PCR
c. Use a spin column to clean up the PCR reaction product
d. Learn how PCR can be used to quantify gene expression

Introduction. PCR amplification of EGFP insert DNA.

PCR reactions use repetitive heating-cooling cycles to anneal EGFP specific primers to a
plasmid that contains an EGFP gene. This plasmid serves as the template for the initial PCR
reactions. A heat-stable DNA polymerase (Taq) uses the annealed EGFP primers to extend a new

20
copy of DNA from the template. Using a set of primers chosen to anneal at specific sites at either
end of the EGFP gene the Taq polymerase enzymatically generates many copies of the EGFP
gene, as illustrated in figure 10.
Figure 10

https://en.wikipedia.org/wiki/Polymerase_chain_reaction

Protocol for PCR.

1. To illustrate the ability of PCR to amplify useable amounts of DNA from trace amounts
of starter template DNA you will set up a series of PCR reactions with pEGFP-N1
plasmid template diluted 1:20, 1:50, 1:100, and 1:500.

2. Record the amount of input plasmid DNA at each of these dilutions (original nanodrop
quantitation).

3. To set up these reactions you will use the p10 pipetman in order to accurately measure
the small volumes.

4. PCR Reactions
a. Set up the PCR reactions with each of the four dilutions of pEGFP-N1

b. Use a small thin-walled 8-tube PCR strip for assembling up to 8 individual reactions

c. To each tube add the following:

(i) 10 ul of 2X Taq buffer & polymerase enzyme mix - (held on ice)
(ii) 8 ul of dH2O to achieve a final volume of 20 ul
(iii) 0.5 ul each of the two PCR primers; EGFP-Nco I and EGFP-Not I primers

21
 (iv) 1ul of the diluted plasmid DNA (the template)

d. Carefully and delicately, securely seal the lids on the tubes of the 8-tube strip and
number in small print. Record the sequence in your lab book.

e. As a control for non-specific amplification set up a PCR reaction without any

pEGFP-N1 plasmid template in the reaction.

f. Set up another control without the primers.

g. If you wish set up a third control with pUC19 plasmid DNA (no EGFP gene)

h. The tubes will be placed in a PCR machine that runs the cycles of heating/cooling.
The program 2015SWG runs 30 cycles of denaturing/annealing/extension.

i. At the completion of this PCR program the reactions will be removed to a refrigerator
until the next lab. In the next lab session you will clean up the EGFP PCR product by
using a spin column and you will evaluate the specificity and abundance of the PCR
products on an agarose gel.

Day 4: Analysis of PCR gene amplification:

Analysis of the PCR amplified pEGFP-N1 insert
You will use an agarose gel to determine whether the reactions in the PCR tubes containing the
four dilutions of pEGFP-N1 from the previous lab have gone to completion.

1. Prepare a 1 % agarose gel just as you did on Day 3.

2. While the gel is solidifying, prepare samples for analysis. To each of four tubes add:
a. 1 ul from one of the PCR reaction tubes (i.e. each tube gets an aliquot from a
different PCR reaction)
b. 8 ul of TE
c. 1 ul of 10X blue dye

3. Once the gel has solidified, load all 10 ul from each of the four tubes onto separate wells
of the gel.

4. Also, in a different well, load a 5 ul aliquot of the 1 kb ladder to estimate if the DNA
insert that you amplified from the plasmid runs at the predicted size.

5. Start the electrophoresis run. Remember: slow migration = better resolution

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Note: Either while the gel is running or after you have run the spin column clean-up of the PCR
DNA (see below), you will participate in a demo lab of quantitative PCR. qPCR is a method for
determining the relative expression of genes in cells or tissue.

Cleanup of the amplified pEGFP-N1 insert using a spin column

After you have determined if the digestions are complete you will use a silica spin column to
remove enzyme, salts, and inhibitors from one of the PCR amplified samples. These DNA clean
up columns work on the same principal of silica binding and elution similar to the small-scale
plasmid isolation spin columns. The reason for doing this is that in the next lab you will be using
the PCR amplified DNA to make ligands and analyze them on a gel

1. Based on the results you have obtained from the agarose gel in the previous section,
select the PCR reaction that you feel has given you the largest yield of the amplified
pEGFP-N1 insert. [Note: you may want to discuss this choice with your TA]

2. The general spin column protocol is shown in figure 11 below.

Figure 11

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 3. Procedure for running the spin column. Note: All centrifugation runs are at 6,000
rpm
a. Add 125 ul of buffer PB to your EGFP PCR product and mix.
b. Place the column in the collection tube.
c. Add the sample to the column and centrifuge for 30 seconds
d. Discard the flow through
e. Wash the column and bound DNA by adding 750 ul of buffer PE, centrifuge for
30 seconds
f. Discard the flow through and re-spin the column for 30 seconds, discard any
residual flow through
g. Transfer the column to a new micro centrifuge tube.
h. To elute the DNA add 50 ul of buffer EB, hold for 1 minute and centrifuge for 1
minute
i. Transfer the eluted, cleaned up DNA to a new tube with a lid.

This column purified EGFP DNA will be used in the DNA ligation lab exercise. Store the
recovered digested plasmid DNA at -200 until quantified by Nanodrop.

For general discussion and follow up in your lab report

What is special about the Taq DNA polymerase?

What is the size of the EGFP PCR product?
Based upon your nanodrop quantification of the EGFP PCR product and your dilutions
how much template DNA resulted in efficient PCR of the EGFP DNA cassette, that is
how sensitive is a PCR assay (ng, pg, or even fg amounts)?

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 Lab Activities (Days 5 & 6)
Overview
The fifth day of lab activities in this Module is broken down into three parts:
1. A set of experiments in which you will ligate various samples of DNA to obtain higher
molecular weight varieties
2. Experiments in which you will isolate genomic DNA from your own cheek cells
3. Restriction digestion of the genomic DNA and analysis of the fragments

Goals
The goal of this laboratory is for you to become competent at performing ligation (covalent bond
formation) of linear DNA molecules. This linking of new DNA, typically joins a segment of
DNA that you wish to isolate to a plasmid digested with a restriction enzyme. Ligation is the
final step prior to transforming re-formed DNA molecules into a bacterial host for identification
of the desired plasmid-plus-insert recombinant molecule. To learn about how to generate
recombinant DNA and monitor success you will set up ligations using the most reliable and
common form of the enzyme- T4DNA ligase.

(Note: there are also other techniques that accomplish the cloning by recombination but this is
a methodology that lies beyond the range of techniques used in this course.)

Introduction to DNA ligations

Successful ligations depend on four parameters: (i) the quality of the cohesive matching ends of
the DNAs, (ii) the type of ligase, (iii) the ratio and concentration of DNA, and (iv) the reaction
temperature. As shown in figure 12 below, DNA ligase catalyzes bond formation between the 3
hydroxyl and 5 phosphate on DNA completing the phosphodiester link.

General properties and guidelines for the enzyme T4 ligase

T4 ligase very efficiently forms the phosphodiester bonds by consuming ATP. T4 ligase works
most effectively under reducing conditions. Thus, all buffers for ligation will include ATP and
the reducing agent dithiothreitol (DTT). Most commercial ligases are concentrated and supplied
as several units per microliter. For T4 ligase 1 unit joins 50% of 1ug of cohesive ends in 30
minutes. We will use the Cadillac of all ligases that is 400 units per ul. Therefor even 1 ul of our
ligase preparation is many hundred-fold excess of enzyme required for typical cloning, thus
placing the odds in your favor.

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Figure adapted from Molecular Cell Biology, Lodish et al, 5th edition

Day 5
Ligation Reactions and controls:
Your team will set up a series of ligation reactions and assess the extent of completion on
agarose gels. The three DNAs to be used include:
1. the 1 kB ladder DNA that you have been using as size markers in previous agarose gels
2. the EGFP PCR product that you prepared and cleaned up in the experiments of days 3 &
4
3. left over restriction digested pEGFP-N1 plasmid

A very useful control to assess your reagents is to set up ligations of the 1 kb ladder DNA. This
is ideal in the setting of our learning laboratory. The DNA ladder is at a known concentration.
The range of sized molecules in the ladder permits easy visualization of the ligation process as
the appearance of new species of larger bands and higher molecular weight forms of DNA higher
in the gel.

In addition to the control ligation using the 1kb ladder DNA, you will ligate two experimental
DNAs, one using your EGFP-amplified DNA and the other using the linearized plasmid DNA
that you prepared as part of the amplification process.

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Protocol for the ligations. Each of the three types of DNA that you will be ligating also includes
a control reaction that is done in the absence of the ligase. The reactions without ligase serve as
a negative control to evaluate if there is any nuclease activity (DNA degrading) or inhibitors in
your reagents and as a negative control when you proceed to use your ligations for bacterial
transformation.
You should also note that the use of small reaction volumes (10-20 ul) are best so that
the ends of the molecules are concentrated. Because T4 DNA ligase is very active you
should set up the reactions on ice.

1. Label the tubes as follows

DNA ladder + ligase

DNA ladder ligase
EGFP PCR + ligase
EGFP PCR ligase
Not I plasmid + ligase
Not I plasmid - ligase

2. Set up the following six reactions (20 ul final volume). Note: If the yield of your PCR or
plasmid recovery was low you can increase the amount of DNA based upon your
Nanodrop readings to obtain >100ng and reduce the amount of dH20 so that you still have
a 20 ul reaction volume.

DNA Ladder ligation

dH20 13ul 12ul

10X ligase buffer 2ul 2ul
DNA 5ul 5ul
Ligase -- 1ul

EGFP PCR product ligation

dH20 15ul 14ul

10X ligase buffer 2ul 2ul
DNA 2ul 2ul
Ligase -- 1ul

Not I digested pEGFP-N1 plasmid ligation: (IF AVAILABLE)

dH20 13ul 12ul

10X ligase buffer 2ul 2ul
DNA 5ul 5ul
Ligase -- 1ul

3. Once you have set up the reactions remove them from ice to room temperature for 30
minutes.

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 4. Prepare a 1% agarose gel to analyze the ligation reactions.

5. After 30 minutes of ligation, add 2.5 ul of 10X loading dye to each reaction tube, mix and
load 15-20 ul on the gel. Run the gel at 70 volts~ 30 minutes.

6. PHOTOGRAPH your results. You should see a shift up (increasing size) of the discrete
ladder bands into high molecular weight ligated molecules.

Day 6
Isolation of mammalian genomic DNA
Oral epithelial cells are easily collected and will be used as a cellular source for lysis and
isolation of mammalian DNA. The size and complexity of this representative high molecular
weight DNA will be evaluated by agarose gel electrophoresis of undigested DNA and restriction
digested DNA.

Cell Harvest A modification of the oral swab technique used during module one will be used to
harvest cells for this activity.
1. Add 500 ul of cell suspension buffer into a 1.5 ml micro centrifuge tube.

2. Use a sterile cotton swab and vigorously swab the lining of both sides of your cheek.
Insert the tube into the 1.5 ml tube and twirl the swab rapidly for 30 seconds to release
the cells adhering to the swab.
The cell suspension should be visibly turbid (cloudy) compared to the buffer by itself. If
the tube is not cloudy you can scrape again with a second swab and add these cells; you
need to twirl the swab quickly.

Cell Recovery
3. Gently pellet the cells by low speed centrifugation of your cell suspension (use a balance
tube) at 5,000 rpm for 2 minutes.

4. Carefully remove the centrifuge tube and inspect the tube. The cell suspension fluid
should now be clear and a visible tiny tear-drop shaped pallet should be at the bottom
outside edge of the tube. This small translucent pellet are your cells.

5. Slowly pipet off the fluid overlay (supernatant) without disturbing the cell pellet.

6. Wash the cells by resuspending the pellet in fresh 0.5 ml of suspension buffer. Do this by
gentle pipetting, or shut/cap the tube and flicking the tube, but no vortexing, not even a
bit.

7. Pellet the cells a second time by centrifugation at 5,000 rpm for 2 minutes. Verify a cell
pellet is present and again aspirate off the supernatant.

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Cell lysis. You will lyse the cells by addition of an extraction buffer containing detergent,
proteinase K, and concentrated buffer and salt.
8. Add 0.5 ml of the cell lysis buffer to the tube, pipet gently to resuspend the cells and
begin their lysis. Cap the tube and invert the tube 10-12 times

9. Place the tubes in a 55-600 incubator for 20 minutes.

Recovery of DNA

10. Remove the vial from the 550 oven and cool to room temperature, 3-5 minutes.

11. Add 500 ul of absolute EtOH to the vialadd the ethanol slowly as an overlay and cap
the tube.
You will see a cloudy interface form. Then slowly mix the EtOH and aqueous phases by
slow inversion 8-10 times.

12. Hold the vial overhead as you mix the phases; you will see a tiny translucent cotton-like
precipitate that slowly condenses to a small dense white mass. This is the high molecular
weight genomic DNA that you extracted from the cells.

13. Centrifuge in the table top centrifuge at 6,000 rpm for 2 minutes. To assist in
locating/visualizing the DNA pellet place the microtube in the centrifuge with the hinge
facing outward. The DNA (near invisible) will be at the bottom on the hinge side.

14. Slowly pipet off the supernatant without disturbing the DNA pellet.

15. Add 250 ul of absolute EtOH to the DNA pellet, cap the vial and invert 2-3 times.
Centrifuge again and remove the EtOH wash. With a p200, pipet off the few microliters
of EtOH that collect over the pellet. Air dry the pellet.

16. Add 100 ul of buffer T.E. Cap the vial and flick the vial to loosen the pellet and begin to
dissolve the DNA. This will take 5-10 minutes. IF the resuspension is partial, open the
vial and very gently pipet the DNA-buffer slowly to complete the DNA dissolving in the
buffer.

17. If time permits set up the restriction digests for gel electrophoresis, if time is short do not
prepare the gel and place your DNA in the cold room until our next session.

Restriction digestion of genomic DNA and gel:

You will use 5 ul of your genomic DNA for each restriction enzyme reaction (10ul total
volume). You will set up one reaction without any enzyme as a no-digestion control for
intact high molecular weight DNA. You will set up three restriction enzyme digestion
reactions with three different enzymes; NcoI, NotI, and XbaI.

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 1. For each of the 4 reactions add:

5 ul genomic DNA
3 ul dH20
1 ul of the 10x restriction enzyme buffer
1 ul of the restriction enzyme

2. Mix by pipeting and place the reaction tubes in a 370 water bath for 20-30
minutes (depending on available time).

3. Prepare a 1% agarose gel with EtBr as you did in the previous days lab

4. Add 1.2 ul of the 10x blue loading dye to each restriction digestion and mix by
pipeting.

5. Record the order that you will load the reactions on the gel.
a. Load 3 ul of the DNA ladder in the first well
b. then your non-digested genomic DNA
c. then your 3 restriction digests
d. then the 6th well with another 3 ul of DNA ladder so that there are size
markers on either side of your DNA digests.

6. Turn on the power and run the gel at 75V for 15-50 minutes or until the blue
tracking dye is at least half way down the gel.

7. Remove the gel to a carrying tray and transport the gel to a UV light box, place a
ruler alongside the gel and photograph the gel under UV illumination.

TO INCLUDE IN LAB REPORT:

Observe the difference in the size of intact genomic DNA relative to the ladder
size markers.
Note the effect of genomic restriction digestion vs plasmid DNA digestion.
Discussion: What accounts for these differences?

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