PCR (Polymerase Chain Reaction) + Gel Electrophoresis Explained

By: Timothy Tong and Grace Suk (edited JL)

Purpose: to amplify a specific DNA sequence and visualize it on a gel

Necessary reagents for PCR
- the DNA sequence to be amplified
- the 4 dNTPs (deoxynucleoside triphosphates): dATP, dCTP, dGTP, dTTP

PCR Buffer that may or may not contain MgCl2

MgCl2
A forward primer and a reverse primer
A thermostable DNA polymerase
PCR grade water

How PCR works

- Generally
o Each cycle of PCR denatures the double stranded DNA molecule of interest,
attaches a forward primer to one strand and a reverse primer to the
complementary strand, and strings dNTPs together to form the complementary
strands to the two original strands. This process is cycled through many times to
amplify even the smallest trace of DNA in an exponential manner. Generally, if
PCR were to run through n cycles, the number of DNA molecules formed would
be 2n.

- More specifically
o When all the necessary reagents are combined, PCR begins when the mix is
heated to 98 C to activate the thermostable DNA polymerase (in our case, Taq
polymerase). This is called the initialization step.
o Each cycle then begins by heating the mixture to 94 C - 98 C to denature the
two DNA strands. This occurs because the hydrogen bonds between the two
complementary strands (or more specifically, between the base pairs of the two
strands, adenine/thymine and cytosine/guanine) are highly sensitive to
temperature and break easily when the temperature is too high (their melting
temperatures are called Tm). This step is called the denaturation step.

o Next, the temperature of the mixture is lowered to 5065 C so that the primers
can anneal to the denatured template DNA strands. The forward primer anneals to
one DNA strand where it is complementary and the reverse primer binds to its
complementary DNA strand where it is complementary. Taq polymerase also
binds to the template DNA strands as well. This is called the annealing step.
o The temperature of the mixture is then raised to the temperature of optimal
activity for the DNA polymerase used. In our case, the temperature of optimal
activity for Taq polymerase is approximately 72C. At this temperature, Taq
moves along the single-stranded template DNA strands in a 3 to 5 direction,
thereby synthesizing the new complementary strands in 5 to 3 manner. Taq does
this by recruiting free floating dNTPs as it moves along the template strands and
stringing them together in a polymerization reaction that yields PPi
(pyrophosphate). In cells, this is hydrolyzed to release a lot of energy (this
reaction, catalyzed by pyrophosphatase, makes DNA polymerization a very
energetically favorable process). The MgCl2 promotes DNA/DNA interactions and
forms complexes with the dNTPs that are the actual substrates for Taq
Polymerase. Without MgCl2, Taq cannot use the dNTPs and DNA polymerization
would not occur. This step is called the extension/elongation step.

o The temperature of the mixture is then maintained around 72C to ensure the
complete replication of the DNA template strands. This is the called the final
elongation step.
o The cycle is now complete, and the next cycle begins again by heating the
mixture to 94 C - 98 C in the second denaturation step. When all the cycles have
been completed, the PCR product should be stored at -4C to preserve the DNA
and prevent its degradation.
o To determine if the PCR worked, gel electrophoresis can be performed.

How to do PCRs in the Bowman Laboratory + explanation (specific to Dirofilaria

Immitis)
- Jans Tips and Tricks
o Do not get distracted while doing PCR. Focus!
o Do not start PCR after 2pm. If you are starting around then, ask Jan when shes
closing the lab that day. Give yourself (and Jan) enough time for PCR to run
(around 2.5 hours)
o Dont let the Thermocycler run overnight or for an extended period of time at 4C
its hard for the machines to maintain that temperature.
o Never trust anyone elses work. If someone else is just finishing their master mix,
dont assume they used proper protocol during their PCR. Take the time to
decontaminate everything again yourself.

- Fill the ice box with ice (located in the hallway) and place the PCR reagents (dNTPs,
PCR grade water, primers, MgCl2, etcexcept for the Taq polymerase take that out just
before you add it to the master-mix to prevent degradation at room temperature) on the
ice so that they thaw out gradually. Do this step first because the reagents take a while to
thaw out. These reagents are located in the left 20C freezer.

- Allow the reagents to thaw completely. Periodically mixing the reagents on clean
vortex will accelerate this process (there should be a new Kimwipe on the vortex). This
step is important for uniform concentration.

- Before you start,

o Fill out the PCR protocol (can be found online). If you have more than 10
samples, be sure to add 1 to the number you scale the original concentrations by
to determine the total amount of each reagent to add, so that you will not run out
of master-mix to add to the PCR tubes.
o Obtain a 1.5 mL microfuge tube for master-mix (preferably a colored one, so it
can be seen on the ice, found in microfuge tube drawer) and one 0.2mL PCR
tube per sample (found in electrophorese drawer under PCR drawer). Label these
PCR tubes according to the sample given on top of the PCR label box (labels are
found on the shelf next to the shelf with the project binders/notebooks).

- Put on the PCR lab coat (near the front door of the lab). Get a diaper (found in a
drawer labeled Diapers and leave it on the PCR area without opening it).

- UV Irradiate the PCR pipettes, PCR tubes, pen and the master-mix tube on the 5th
floor in C5 168A. Irradiating eliminates DNA contamination by cross linking DNA
eliminating false positives due to contaminated supplies. In order to irradiate, open the
PCR tube rack cover to irradiate the inside note all tubes should be open to allow UV
to penetrate inside of tubes. Choose Autocross and then Start. Flip the pipettes and
the pen and run autocross again. Leave the tubes inside the machine. Change gloves to
make sure you are not touching the irradiated items with the same glove you touched

them with before irradiation and put the cover back onto the PCR tube rack, close the
master-mix tube lid, cover the PCR tubes with rack cover (leaving PCR tubes open)and
put the pipettes and the pen in the ziplock bag.
o It is important to distinguish between PCR pipettes and other pipettes, because
PCR pipettes are supposed to be free from any sort of contaminant DNA that can
also be accidentally amplified by PCR.
o The irradiated pen will be used to cross off reagents added on your PCR protocol,
so you can keep track of what was added and what wasnt.
o When you are walking through the hallways, you should only be wearing one
glove. Use your gloved hand to carry the pipettes, the pen (within the ziplock
bag), PCR tubes and master-mix tube (in a PCR tube rack) and used your
ungloved hand to open doors.

- Turn on the PCR cycler (make sure it has been running for at least 30 minutes before
you start the PCR, to make sure the machine is warmed up). Close the lid during warm
up so the heat doesnt escape and cause the thermocycler to overheat.

- Open the diaper and take out the pipettes you will need.
- Using PCR pipette tips, add water first, then PCR buffer, and then rest of the
reagents. Vortex the master-mix when finished. Use a new pipette tip for each reagent.
o Remember to vortex each of the reagent tubes before adding to the master-mix
tube to ensure sufficient mixing (a new Kimwipe, different from the one used to
thaw out the reagents, should be used) and to be positive you are adding a wellmixed reagent, use your pipette to mix before you remove reagents from the
reagent tubes (to do this, repeatedly press and release the pipette button, so that
reagent is repeatedly being extracted and re-pipetted into the tubeyou should
see the level of the reagent changing slightly; this essentially mixes it).
o Reminder to pipette small volumes directly into the existing mix. Ex: when
adding the primers, put the pipette tip into the rest of the mix.
o Minimize contamination by closing the lids after each addition.

- Open the PCR tube rack, keeping the cover facing down. Using a PCR pipette and PCR
pipette tips, add the master-mix into each PCR tube (negative control last, because
since it is not supposed to come up positive, it is okay if it does not have as much mastermix as the other samples). Do not lift up the tubes leave them in the rack. Start with
the bottom tube to minimize reaching over and contaminating the other tubes. Put the
rack cover back on.

- Put away all the reagents, PCR pipettes, and tips. Take off the PCR coat. Take out your
DNA samples from the freezer and allow them to thaw out, and lightly vortex. These
samples should be located in the right 20C freezer.

- Using DNA filter tips in the electrophoresis drawer and a regular pipette, add the DNA
samples into their corresponding PCR tubes (try to do this away from the PCR area, so

that no DNA can get into future PCR reactions). Again, start with the bottom tube, but
this time, lift them up and dip the pipette tip in the master-mix to make sure the DNA
sample is added into the solution. Close the lid. Do not add anything in the negative
control. Close the negative control tube last. Once again, remember to mix by gently
vortexing.

- Put the PCR tubes into a PCR Thermocycler (there are two). Press Start Enter (no
need to enter actual date but just press enter) Program Yes (for using existing
program) Enter program number Make sure to use correct program with correct
conditions) Start. Make sure the lid is closed. Put a label on the lid (D. immitis,
Date, Initials) so that Jan or someone else working in the lab can remove your PCR
products and place them in fridge after you leave.

- Throw away the master-mix tube and put the used diaper under the fecal floatation
centrifuge next to the hood so that it can be reused for non-PCR experiments.

Works Cited
Blicq, D.Nucleic Chemistry. 4th January 2010. 15th May 2012. http://xnet.rrc.mb.ca/davidb/nuc
leic_chemistry.htm
DNA Repair and Replication. 15th May 2012. http://web.virginia.edu/Heidi/chapter30/chp30.htm
Polymerase Chain Reaction 6th May 2012. Wikipedia. 15th May 2012.http://en.wikipedia.org/w
iki/Polymerase_chain_reaction

How gel electrophoresis works

- Generally, gel electrophoresis separates DNA molecules by size. DNA is a negatively

charged molecule (due to the negatively charged phosphate groups) and gel
electrophoresis exploits this by creating an electric field using a positively charged
electrode on one end and a negatively charged electrode on the other end. Because
opposite charges attract, DNA tends to move towards the positively charged electrode in
a manner dependent on its size. The gel in this lab is made out of agarose, which is
basically a microscopic maze through which DNA molecules must navigate through to
reach the positively charged electrode. The larger DNA molecules move slower through
the matrix within the agarose because their size impedes them and so are found closer to
the negatively charged electrode (their original location) when the electric field is
removed and the smaller DNA molecules move more quickly because their small size
allows them to navigate through the matrix easily and so are found closer to the
positively charged electrode. The sizes of the DNA fragments can be determined by
utilizing DNA ladders, which come in various sizes. These ladders consist of a solution of
DNA molecules of different lengths, determined by the producer of the ladder. By
comparing the band composed of the DNA of interest to the ladder, the size can be
approximated and if the size corresponds to the DNA sequence to be amplified, the PCR
worked; furthermore, the brightness of these bands can implicate relatively how much
DNA product is in each band; the brighter the band, the more PCR product is contained
within. Further confirmation of the PCR working can be obtained if the PCR products are
sent to be sequenced. Visualization is accomplished by using ethidium bromide, a type of
DNA stain, which binds to DNA so that the bands can be visualized using the ultraviolet
light emitted by the transilluminator.
An example of a completed gel:

How to do gel electrophoresis (25 mL gel at 2% agar) in the Bowman Laboratory +

explanation
- Get the gel electrophoresis Horizon brand apparatus (located in the cabinet under the
printer), place it under the hood and set it up. The two black bars with arrows on them are
the gel barriers and should be placed in their slots, with the arrows pointing inward, and
one comb should be placed in its slot, closer to the side with the black colored electrode.
o Determine the well comb size based on the number of samples and volume
(typically 14)

- The agarose gel is prepared by the following procedure:

o Obtain 0.5 g of agarose from the electrophoresis drawer and pour into a small
bottle with a black cap.
o Obtain 25 mL of 0.5x TBE and pour into the bottle using the 25 mL pipette.
Keep the pipette in the wrapper for later use in pouring gel.
o Place the bottle into the beaker with blue tape saying For Gel and fill the
beaker with water so that the water level rises slightly over the TBE+agarose level
o CAP LOOSELY TO AVOID EXPLOSION
o Microwave the beaker for 1 minute. The microwave can be found in C4 102A.
Remove the bottle and mix it by twirling it around. Place the small bottle back
into the beaker and microwave it again for 30 seconds. This should be sufficient
to dissolve the agarose, but if there are visible agarose chunks remaining,
microwave it again. The solution should be clear.
o Under the chemical fume hood, add 0.625 uL of ethidium bromide (EtBr). Use
10 uL unfiltered pipette tip because it has a shaft guard to prevent contamination
of the pipette. EtBr compound is carcinogenic, so be sure to use gloves and
change them every time you come into contact with it. Dip the tip into the
solution to make sure EtBr is added.
o Mix the contents in the bottle (agarose+TBE+EtBr) by twirling the bottle.
o Using the 25 mL pipette from before, extract 21-22 mL from the small bottle and
carefully pipette it into the gel tray with the comb. Try to avoid making large air
bubbles in the gel and bubbles between the gel and the gel deck. Do not pour from
bottle all 25 mL to avoid making bubbles.
o Afterwards, pour the hot water within the beaker into the bottle and add a cup full
of bleach using a Dixie cup underneath the fecal floatation centrifuge, and after
mixing the contents, pour into the beaker under the hood containing labeled as
EtBr + bleach. This step is done to prevent any residual gel solution from
solidifying in the bottle.
o Let the tray sit for around 25 to 30 minutes until agarose solution solidifies.
o While you wait for the gel to solidify, thaw out PCR products at room
temperature. Print out a gel record sheet and fill in the table. Prepare a sheet of
parafilm and label it with sample numbers.

o Add 3.75 uL of EtBr to the gel electrophoresis running buffer (located in a

bottle saying Recycled TBE running buffer) and enough 0.5x TBE so that
there is a total of 150 uL of running buffer in the bottle (this is an
approximation just use the lines on the bottle, no need to use a graduated
cylinder).

- After the gel solidifies, remove the comb first (carefully! be gentle to avoid tearing the
gel), and then the two black bars. Then pour the running buffer into the gel tray,
making sure to fill every well with it.
o The running buffer should only be used up to 10 times. After the 10th time it is
used, new buffer must be made.
o The function of this buffer is to maintain the pH within the gel tray and contains
the ions necessary to conduct electric charge so that the reaction within the gel
tray occurs smoothly.
o You do not need to add this ethidium bromide in the hood, because the solution is
not hot.
o As always, change gloves every time after dealing with EtBr.

- Now, you can load the wells. First, load 5 uL of 50bp ladder (already contains loading
dye). Combine 5 uL of each PCR sample with 1 uL of orange (6x) loading dye on the
parafilm in their corresponding spots and mix using pipette. Load all your samples (PCR
product + loading dye), making sure you keep track of which well contains which
sample. Afterwards, load another well with ladder, so that one ladder flanks the samples
on each side.
o It is okay if samples leak out from the top of the well into the running buffer. Gel
electrophoresis will only work for the samples contained within the gel.
o Be careful not to puncture the bottom of the gel with your pipette.
o The loading dye serves the purpose to simplify DNA gel electrophoresis in several
ways. One is to allow visualization of the DNA mixture during loading and while
the gel is running in the gel. Loading dye is a mixture of two dyes (blue and
yellow) The yellow color is of small molecular weight and runs in the gel before
any DNA the blue is a heavier weight to show maximum traveled distance.
Loading dye chemical composition assists the DNA to sink to the bottom of the
wells.

- Make sure the gel is centered in the tray and also ensure that the gel tray is relatively flat
on the table. Do this by adjusting the heights of the legs of the gel tray by rotating the
wheels at the bottom of the legs to center the air bubble in the compass as much as
possible.

- Hook up the gel electrophoresis tray to the EC apparatus so that the colors of the wires
correspond to the colors of the ports. Turn on the apparatus and run the gel for 45
minutes at around 90 V. Make sure you see bubbles rising near the black colored
electrode to ensure that the apparatus is actually running.
o To do this, make sure the power switch on the clock is turned on.

o Make sure the power button on the EC apparatus under the clock is turned on (I is
on, and O is off).
o Rotate the black arm on the clock to 45 minutes to start the clock.

- After 45 minutes, remove the gel and place it in container saying For Ethidium Bromide
Gels to visualize in the gel electrophoresis visualizing room on the 5th floor, C5 148.
Bring gloves and several paper towels.

- In the gel electrophoresis visualizing room,

o Make sure you are wearing gloves. Assume that everything in there is coated with
EtBr even the computer.
o Wipe down the black platform on which you will be placing your gel with water
and a paper towel.
o Place your gel in the center.
o Turn on GeneSnap, change the settings to upper white light, press the green
button, and position the gel as center as possible and zoom in as much as
possible (the green button will have turned red).
o When you have reached the optimal visualization position, close the door of the
apparatus completely, turn on the Transilluminator UV light, and then press
the green button again (the green button will have turned red again).
o Change the exposure time from 34 ms to 250 - 300 ms. You should now see the
gel (colored black) and glowing white bands (of the ladder and your samples).
o Press the red button to adjust the lighting/contrast etc. on the right side so
that the gel can be clearly interpreted.
o Print a picture of the gel for your records and take off one glove to tear it
from the printer.
o When youre finished, remove the gel and wipe down the platform with water and
a paper towel.

- Back in the lab, place the gel into the beaker under the hood containing bleach to dissolve
it and pour the running buffer within the gel tray back into its original bottle. Indicate that
you have used the running buffer by adding one tally to the label on the bottle holding
running buffer.
o If there is not enough bleach in the beaker, add more.

- Finally, attach the photo to your gel electrophoresis recording sheet and analyze your
results.

Works Cited

The Basics of Gel Electrophoresis Alaska BIOPREP University of Alaska Fairbanks. 15th May
2012. http://bioprep.wikis.uaf.edu/The+Basics+of+Gel+Electrophoresis
Blicq, D.Nucleic Chemistry. 4th January 2010. 15th May 2012. http://xnet.rrc.mb.ca/davidb/nuc
leic_chemistry.htm