LAB 6:
Agarose Gel Electrophoresis of
Digested DNA & PCR Set-Up
I. Objectives
The purpose of today’s lab is to learn how to set up and run an agarose gel, separate DNA fragments
on the gel, and visualize the DNA on a transilluminator. Students will also learn how the polymerase
chain reaction works, how to design PCR primers, and how to set up a PCR reaction. In addition,
students will learn how to determine the concentration of DNA in a DNA extract. By the end of today's
lab, students should:

Know how to pour, load, and run an agarose gel

Be able to interpret the DNA bands observed on an agarose gel

Be able to explain how the polymerase chain reaction (PCR) works

Know the role of each of the reagents in a PCR reaction

Be able to explain what factors need to be taken into consideration when designing
primers for PCR

Know how to set up a PCR reaction and load it into a thermocycler

Be able to estimate the amount of DNA in a DNA extract
II. Safety considerations

WEAR GLOVES! Ethidium bromide has been added to the gel buffer and the agarose. It is a
powerful mutagen and can cause mutations in skin cells it touches. NEVER HANDLE THE GEL
BUFFER, AGAROSE OR GEL RIGS WITH YOUR BARE HANDS!!!
III. Introduction
A. THE POLYMERASE CHAIN REACTION (PCR)
The polymerase chain reaction (PCR) is a technique for copying or "amplifying" DNA in a test tube.
Discovered by Kari Mullis at Cetus Corporation in the 1980s, it revolutionized the field of molecular
biology and won Mullis a Nobel Prize in Chemistry in 1993.
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The power of PCR lies in its ability to make billions of copies of a specific, small
DNA sequence of interest. Consider the following analogy. Imagine that you
have a pile of a thousand pennies. Among that stack is a particular penny of
interest that is very valuable and that you want to study further. However, to
study that penny you first need to find it among all the other pennies in the
stack. That could be a very tedious task without some type of tool to help you out. Now imagine that
you have a “penny copying machine” that can somehow seek out the special penny and make a billion
copies of it. The machine doesn’t isolate the special penny you’re after from all the other pennies in
the stack. However, it makes so many copies of it that you now have a huge stack of special pennies
to study and the stack contains only a very small percent of pennies that are ordinary.
Where PCR is concerned, genomes are like stacks of pennies. The “pennies” are DNA sequences.
However, a researcher is usually interested in studying only one or a handful of DNA sequences at a
time (e.g. the human sickle cell anemia gene, the regulatory region of a bacterial gene involved in
glucose metabolism, or polymorphic markers used for human identification). PCR is like the “penny
copying machine” because it makes copies of a specific DNA sequence. The PCR reaction doesn’t
eliminate the rest of the DNA but, rather, it overwhelms it by making billions of copies of the sequence
of interest. Thus, the researcher has plenty of his or her specific DNA sequence to study and
encounters only minimal interference from the rest of the DNA (the “ordinary” pennies).
PCR works by mimicking the replication of DNA that takes place in cells. However, rather than
producing two identical copies of the entire genome (as a cell does prior to dividing), PCR produces
billions of copies of small (usually 2,000 base-pairs or less), highly specific stretches of DNA.
The PCR reaction involves 4 steps: (1) denaturation, (2) primer annealing, (3) primer
extension, and (4) cycling. The molecular events happening at each step are described below and
the entire process is illustrated in Fig 9-3. Note that the end result is many copies of a specific small
segment of DNA defined by the sequences of the primers that are used in the reaction.
STEP 1:
Denature Template DNA. In order to replicate the DNA, it must first be denatured
(made single stranded) to expose the strands to the cellular enzyme that replicates DNA,
DNA polymerase. In cells, enzymes called helicases denature the double-stranded DNA. In
PCR, heat is used to accomplish this task.
The reaction tubes are raised to 95 degrees C for about one minute to denature the DNA.
It is critical that the template DNA be completely denatured.
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STEP 2:
Anneal Primers. Once the DNA has been denatured, the single-stranded DNA primers will
find their complementary sequences and hybridize, or anneal. The primers are required to
specify to the DNA polymerase which region of the DNA is to be copied and to provide the
free 3’-OH groups required by DNA polymerase to start synthesizing a new DNA strand.
The annealing temperature depends largely on the sequence of the primers. For greatest
specificity, primers are annealed at or near their melting temperature – the temperature
at which the primer will just barely hybridize with its complementary sequence without
melting off. Since A-T base pairs are less stable than G-C base pairs, A-T rich primers
have lower melting temperatures than do G-C rich primers. To get an estimate of the
annealing temperature of a primer, assign 2° C for each A or T in the primer and 4° C for
each G or C.
What melting temperature would you assign to the following primer:
5’- aac tta agc taa cac cgt agg –3’ ?
Estimated Melting Temperature (Tm): ___________° C
PCR primers are usually about 20 bases in length to ensure specificity. If the primer is too
short, it will anneal to other sequences of DNA that have the sample sequence as the
target DNA just due to random chance.
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For example, if a primer was 5 bases long (e.g. 5’-ggcat-3’), it would anneal to every 3’ccgta-5’ sequence in the target genome. This sequence would be expected to appear by
random chance every 45 base-pairs, which is about once every 1,024 base-pairs. In the
human genome, which has 3.3 x 109 base-pairs of DNA, this sequence would appear over
2 million times. Clearly, a primer that is 5 bases long is not very specific!
If a primer is 20 bases long, it will only appear by random chance every 420 base-pairs, or
once every one trillion base-pairs. Therefore, it should be highly specific in a human PCR
amplification reaction.
STEP 3:
Extend Primers. After the primers have annealed, DNA polymerase recognizes their 3’
ends and begins extending both primers in the 5’ to 3’ direction. Bases are added
according to the standard base-pairing rules, where A pairs with T and G with C. Note
that as this happens, the primers on the two strands are extended toward one
another across the target sequence. They will not “meet” however, since the primers
are being extended on two different strands of the original parent DNA molecule. Since the
most favorable temperature for heat stable DNA polymerases is about 72 degrees C, the
extension step is usually run at about this temperature.
STEP 4:
Cycling. At the conclusion of step 3, the DNA target sequence has been copied one time
to yield a total of two target DNA molecules. By itself, this is not very interesting since the
DNA has only been amplified 2-fold. However, notice what happens when the steps are
repeated a second time: 4 target molecules result. After cycle 3, there are 8 molecules;
after cycle 4 there are 16, etc. In other words, the number of copies of the target DNA
sequence increases exponentially, so that after 30 cycles there are about 230 copies of the
sequence – a billion-fold amplification!
Cycling is made possible in part because a vast excess of primers and dNTPs are added at
the beginning of the reaction. Therefore, the reaction does not stall due to lack of raw
materials. In addition, the thermal stable polymerase eliminates the need to add fresh
DNA polymerase at the beginning of each extension. The heat stable polymerase can
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survive the cyclical denaturation steps (95 degrees) that would permanently destroy the
activity of most DNA polymerases.
A standard PCR reaction requires the following ingredients:
1. Template DNA – This DNA is the source of the DNA sequence that you seek to amplify. In
forensics, the template DNA is usually human. You would obtain human cells from a cheek
swab, a blood sample, etc. and then apply one of a variety of techniques that are routinely
used to extract DNA from intact animal cells. This would then be your template DNA for the
PCR reaction.
2. DNA primers – Within the template DNA is the target sequence you wish to amplify. In
forensics, these are usually 15 polymorphic human STR loci or one of the two human
mitochondrial hypervariable regions. In order to amplify a region of the DNA specifically, you
need to design short, single-stranded DNA molecules called primers. These primers are made
synthetically (they are usually ordered from a company for about 50 cents per base) and are
designed so that they will anneal to the upper and lower strands of the region immediately
flanking the target DNA sequence.
3. dNTPs – Deoxynucleotide triphosphates are the raw ingredients for DNA replication. DNA
polymerase cleaves off two of the phosphates to provide energy for the polymerization
reaction and then adds the appropriate nucleotide monophosphate to the 3’ end of the
growing DNA daughter chain. (The “N” stands for either A, G, C, or T.)
4. DNA polymerase – This enzyme catalyzes the formation of a new strand of DNA using
dNTPS and an existing DNA strand as a template. As mentioned above, in PCR, heat stable
DNA polymerases are used because they can tolerate high temperatures (up to 100 degrees
C) without denaturing. Thermal stable polymerases are isolated from heat tolerant bacteria
that live in thermal vents.
5.
Salts/buffer – DNA polymerases require magnesium as a cofactor so PCR reaction
buffers always contain magnesium as well as a buffering agent (to maintain pH).
TYPICAL THERMOCYCLER PROGRAM FOR PCR:
Step 1
95 degrees C
60 seconds
Step 2
55 degrees C
60 seconds
Step 3
72 degrees C
60 seconds
Step 4
Cycle Steps 1-3, 30 times
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The Polymerase Chain Reaction. (From http://aidshistory.nih.gov/imgarchive/pcr.html)
B.
PRIMER DESIGN
Another aspect of PCR that is important to understand is primer design. Primers must be specific, so
that they will not amplify any regions of the DNA that you did not intend to target. The specificity of
the primer lies in its sequence as well as its length.
The sequence is defined by the areas that flank the DNA region you want to amplify. Remember that
one primer anneals to the top strand (running 5' to 3') and the other anneals to the bottom strand (3'
to 5'), and that the 3' ends of the primers must face each other
For example, imagine that you want to PCR-amplify the short sequence below:
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5' - A A A G G C C A G C C T G T T T G C G C T G T T T A G A G C T T C C T - 3'
3' - T T T C C G G T C G C A C A A A C G C G A C A A A T C T C G A A G G A - 5'
In order to design your primers, you need to imagine the strands as denatured:
5' - A A A G G C C A G C C T G T T T G C G C T G T T T A G A G C T T C C T - 3'
3'
5'
5'
3'
3' - T T T C C G G T C G C A C A A A C G C G A C A A A T C T C G A A G G A - 5'
Then remember that the primers will anneal in the opposite orientation to the strand that they anneal
to. In other words, the primers will have to anneal in the orientations shown.
So the primer annealing to the top strand would have the sequence 5'-AGGAAGCTCTAAACAG-3' and
the primer on the bottom strand will have the sequence 5'-AAAGGCCAGCCTGTTT-3'. Can you see
why?
Primers of this length are sufficient to amplify specific sequences in bacterial genomes because
bacterial genomes are relatively small (on the order of a few million base pairs). Let's see why. Since
there are 4 different bases, a sequence like 5'-CT-3' would be found (on average) about once in every
42 = 16 base-pairs. Obviously, a primer of this size would not be very specific and would hybridize
non-specifically all over a bacterial genome. However, a primer with a 16 base sequence would only
hybridize (on average) every 416 = 4.3 x 109 base-pairs. Sequences of this length are specific enough
to hybridize (on average) only one time in the human genome (which is 3.3 billion bp in length).
However, to ensure specificity, primers of about 20 bases are usually used when PCR-amplifying
human DNA.
A final important aspect of designing primers for PCR is that both primers should have about the
same melting temperature. If they don't, then the one with the higher melting temperature will
hybridize non-specifically at the annealing temperature that prevents the other primer from "melting
off" the DNA. For example, if one primer has a melting temperature of 58 deg C and the other has a
melting temperature of 70 deg C, the annealing temperature used in the PCR reaction would have to
be a little lower than 58 degrees to ensure that the primer with the lower melting temperature doesn't
"melt off" the DNA. However, at 56 deg C (or so), the other primer will hybridize non-specifically since
not all of its bases need to pair in order to attain stability. Primers used in a PCR reaction don't need
to have exactly the same melting temperature, but their melting temperatures should be within a
couple of degrees of one another using the "2 + 4 rule."
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C. CALCULATING HOW MUCH DNA EXTRACT TO ADD TO A PCR REACTION
PCR works most efficiently when just the right amount of DNA is added to the reaction. “Just the right
amount” depends on the origin of the template DNA (genomic, plasmid, a previous PCR product, etc.),
the purity of the DNA, and the type of downstream detection system that will be used to visualize the
PCR products. For our purposes, we want to “seed” our PCR reactions with about 50 ng of template
DNA.
You will be provided with positive control DNA samples with known concentrations. However, you will
need to determine the concentration of DNA in your own experimental extract. There are several ways
to do this but one of the easiest and most common is to measure the spectral absorbance of the
extract at a wavelength of 260 nm, since nucleic acids absorb light at this wavelength.
Absorbance is measured in an instrument called a spectrophotometer. Instructions for how to use
the spectrophotometers are located beside the instruments. Absorbance readings must fall between
0.1 and 1.0 to be accurate, so you will have to dilute your sample to get a good reading.
Once you have made your reading, you can use the following equation to determine the concentration
of DNA in your sample:
Concentration DNA in sample = (50 ug/mL)(A260)(dilution factor)
Once you have determined the concentration of your sample, you will then be able to calculate how to
dilute your DNA so that you’ll be adding only about 50 ng of DNA to your PCR reactions.
IV. Things to Do
PART A. AGAROSE GEL ELECTROPHORESIS OF RESTRICTION ENZYME DIGESTIONS
1.
Put on a pair of gloves. If you have long hair, tie it back.
2.
The gel rig, casting tray, power supply, and gel comb, and TAE running buffer are all at your
bench. DO NOT TOUCH ANY OF THESE ITEMS WITH YOUR BARE HANDS; ALWAYS
WEAR GLOVES.
Also make sure that you know the location of:

Melted agarose in water bath

Graduated cylinders (beside agarose)
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3.
Retrieve your tubes of digested DNA from the front bench. Also get one tube of loading dye
from the front bench.
4.
Set up your casting tray and gel comb according to the demonstration by your instructor.
5.
Once your casting tray is ready, get 50 mLs of melted agarose from the water bath, using one
of the graduated cylinders. Carry it back to your bench and pour it into the tray. If there are
any bubbles on the surface of the gel, pop then gently with a micropipettor tip. (The tip should
then be discarded as biohazardous waste.)
6.
While you are waiting for the gel to cool, add 5 uL of loading dye to each of your restriction
digest reactions.
7.
When your gel has cooled, carefully remove the comb (pulling straight up) and place the gel
in the gel rig. Then add enough 1X TAE running buffer to cover. Make sure that the wells are
filled with buffer and that the gel is well-covered by buffer. (You can confirm this by viewing
the gel from the side.)
8.
Load the samples onto the gel according to your instructor's directions. Make sure that your
pipette tip is below the level of the buffer and right above the well when you load. Use the
dark piece of paper that has provided for you to help you visualize the location of the wells.
9.
When your samples are loaded, add the gel rig lid and connect the leads to the power supply:
black to black and red to red. Turn on the voltage to 100 V. Look for the appearance of
bubbles on each end of the rig to confirm that you have a current. (These bubbles arise from
the hydrolysis of water to hydrogen and oxygen gasses.)
10.
While you are waiting for your gel to run, continue on to Part B. Keep an eye on your
gel, though, and watch as the dyes in the loading dye begin to separate as they move through
the gel at different rates.
11.
The gel should run for about 1 hour. When your instructor tells you it is time, disconnect the
leads, remove the lid off the apparatus, and prepare for viewing your gel on the
transilluminator. Since the lab has only two transilluminators and one camera, you will have to
wait your turn for using them. When it's your turn, carefully remove the casting tray (and gel)
from the apparatus. Hold the gel on either end so that it won't slip off the casting tray as you
carry it to the transilluminator.
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12.
View the gel, using UV goggles if there is no protective covering over the transilluminator.
Take a photo of your gel using the polaroid camera set-up. Your instructor or TA will help you.
Did you see what you predicted you would see?
13.
When you are done viewing your gel, discard the gel in the biohazard bag provided and return
your casting tray to your bench. Discard the loading buffer in the large container provided,
and rinse out the casting tray, gel apparatus, and gel comb with dI water. Then return these
items to your bench to dry. DISCARD YOUR GLOVES AS BIOHAZARDOUS WASTE WHEN
YOU ARE DONE.
14.
Before next lab period, one of the members of each group should make photocopies or
scanned (digital) copies of the gel for the other group members so that everyone has a record
to paste into his/her notebook.
15.
Proceed to Part C of the lab.
PART B. SET UP PCR REACTIONS
Which primer sets you use for PCR (each group will use two primer sets) will depend on the results of
your EMB plating experiment. If you think your experimental colony was a coliform of any kind
(including E. coli), then you should use the coliform-specific primer set and the E. coli primer set. If
you think your experimental colony was NOT a coliform, use the coliform-specific primer set and the
universal bacteria primer set.
If you are confused about which primer sets to use, see your instructor or TA before
proceeding!
1.
Collect the following from the front bench for your PCR reaction set-ups:

1 tube coliform-specific primer set (“CPS”)

1 tube bacterial universal primer set (“UPS”) – if needed

1 tube E. coli specific primer set ("EPS") – if needed

1 tube PCR reaction mix (contains dNTPs, taq polymerase, buffer); labeled "RM"

Your extracted experimental DNA (from last time)

Commercial E. coli DNA

Commercial Enterobacter (non-E. coli coliform DNA)

7 thin-walled PCR tubes (0.2 mL)
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1.
Add 700 uL of distilled water to a 1.5-mL tube. Thoroughly mix your experimental DNA sample
(your extract from last time) and then add 50 uL of the extract to the tube of distilled water.
Thoroughly mix the contents of the tube. What is your dilution factor? _________
2.
Take your tube to one of the spectrophotometers and follow the directions to determine the
concentration of DNA in your sample. If your reading is greater than 1.0, you will need to
dilute your sample further and take another reading. Ask your instructor or TA if you need
help!
3.
When you have an absorbance reading between 0.1 and 1.0, use the equation provided in the
introduction to this lab to calculate the concentration of DNA in your undiluted DNA extract.
5.
You will be adding 2 uL of your experimental DNA to the PCR reactions. Since you want a total
of 50 ng to “seed” the reaction, this means that you need to prepare a dilution of your DNA
extract that is at exactly 25 ng/uL.
6.
Work with your lab partners to figure out how to set up your dilution. The following equation
will help. (Make sure your units are consistent!!)
Vi = [(Vf)(Cf)]/(Ci)
For example, let’s say that you dilute your DNA 1:10 and get an A260 reading of 0.42. From
this, you calculate that the concentration of DNA in your undiluted sample is:
(50 ug/mL)(0.4)(10) = 200 ug/mL = 200 ng/uL
You only need a small amount of DNA for your PCR reactions (see the table below), so 50 uL is
sufficient as a final volume.
Vi = [(Vf)(Cf)]/(Ci) = [(50 uL)(25 ng/uL)]/200 ng/uL = 6.25 uL
So to make 50 uL of a solution of DNA at 25 ng/uL, you would add 6.25 uL of your undiluted
DNA to 43.75 uL of PCR water.
MAKE SURE YOU UNDERSTAND HOW TO MAKE THIS TYPE OF CALCULATION BEFORE
COMING TO LAB.
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7.
Once your DNA is ready, label the sides of your 0.2-mL thin-walled PCR tubes with the number
assigned your group by your instructor. (All your tubes should have the same number of the
side.)
8.
Label the tops of the tubes 1-7. (Each tube will have a different number on the top.)
9.
Be very careful from this point on and understand before you come to lab what you
are going to do!!
The table below shows how to set up your reactions. The first 5 reactions are controls (see if
you can figure out why they are controls) and ALL groups will set them up. The last three
columns are experimental reactions and each group will pick only two out of these
three reactions to do. If you think your bacteria may be a coliform of ANY KIND (including
E. coli), then set up reactions 6 and 7. If you think your bacteria is not a coliform, then set up
reactions 6 and 8.
Remember: Each lab group should set up only 7 PCR reactions total!! Be sure to record
in your lab notebook which reactions you chose to perform.
10.
Then add reagents to the tubes as follows:
Tube #
11.
1
2
3
4
5
6
7
8
PCR reaction mix
38 uL
38 uL
38 uL
38 uL
38 uL
38 uL
38 uL
38 uL
Coliform
primer set
E. coli
primer set
Universal
primer set
Your experimental
DNA
(+) control
coliform DNA
(+) control
E. coli DNA
PCR water
(neg control)
10 uL
10 uL
--
--
10 uL
--
10 uL
10 uL
--
--
-10 uL
10 uL
--
--
--
--
2 uL
--
--
2 uL
--
2 uL
2 uL
--
--
--
--
10 uL
2 uL
2 uL
2 uL
2 uL
--
--
--
--
--
--
Gently mix up and down with your pipettor to mix. Try not to introduce any bubbles as you do
this. If you do introduce bubbles, ask your instructor how to remove them.
12.
Take your PCR reaction tubes to the thermocycler. Your instructor will help you load your
tubes into the instrument. The program we will be running will be given to you in the write-up
for Lab 7.
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PART C. STRUCTURE, TENSE, AND VOICE OF SCIENTIFIC PAPERS
1.
Get out the copy of the primary journal article you found at the library or online this week and
show it to your instructor. He or she will confirm that you have chosen the right kind of paper
for next week’s in-lab discussion.
2.
Before next lab period, look at the CITATION information given at the top of the article’s
first page. This includes the title, authors, institutional affiliations, journal, volume, pages, and
publication date. Sometimes, the journal’s publisher is also included in the citation. Some
journals are published by scientific societies (e.g. American Society of Microbiologists) while
others are published by commercial enterprises. If your paper does not indicate the publisher,
get online and find out what organization or company publishes it.
Record the following information in your lab notebook:
3.
a.
Name of the journal
b.
Organization/company that publishes the journal
c.
Volume number and page numbers of the article
d.
Publication date of the article
e.
Title of the paper
f.
Names of the authors and their affiliations
g.
What “key words” are associated with the research in the paper?
Before next lab period, find the website of one of the authors, preferably the last author
listed. Usually, this individual is the principal investigator (“PI” or leader) of the laboratory
in which the research was performed. PIs determine research priorities, write grants, hire
personnel, present findings at conferences, convene lab meetings, write and review
manuscripts, and are ultimately responsible for the work done in their labs.
Record some of the information you find on the website in your lab notebook. Is the
author a professor at a University or does he/she work for a company? What are the research
interests of the author (in general)? The web sites of most active scientific research lab PIs
have links to their other publications and a brief summary of their research focus.
V. Lab Clean-Up

Make sure your gel tray, comb, and gel apparatus have been rinsed with dI water and have
been placed back on your bench

Discard all leftover tubes from PCR reaction set-ups in the trash
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