Download Polymerase Chain Reaction (PCR) + Electrophoresis with agarose

Polymerase Chain Reaction (PCR) + Electrophoresis with agarose gels
Overview: PCR is a scientific technique in molecular biology to amplify a single or few copies of a
piece of DNA across several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating
and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short
DNA fragments, 10-20bp) containing sequences complementary to the target region along with a DNA
polymerase (after which the method is named) are key components to enable selective and repeated
amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting
in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be
extensively modified to perform a wide array of genetic manipulations.
After PCR of a sample, one technique that is commonly used is DNA electrophoresis.
DNA electrophoresis is an analytical technique used to separate DNA fragments by size. Because you
developed primers to amplify a specific segment of DNA, after PCR you should know the size of the
amplified DNA fragment. DNA molecules which are to be analyzed by DNA electrophoresis are set
upon a viscous medium, the gel, where an electric field forces the DNA to migrate toward the positive
potential, the anode, due to the net negative charge of the phosphate backbone of the DNA chain. The
separation of the DNA fragments from your PCR reaction is accomplished by exploiting the mobilities
with which different sized molecules are able to traverse the gel. Longer molecules migrate more slowly
because they experience more drag within the gel. Because the size of the molecule affects its mobility,
smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the
voltage is removed and the fragmentation gradient is analyzed by comparing the PCR products to a
ladder (DNA products of known size; see below) that is run simulaneously. For larger separations
between similar sized fragments, either the voltage or run time can be increased. Extended runs across a
low voltage gel yield the most accurate resolution.
POLYMERASE CHAIN REACTION (PCR)
Materials:
- Primers (both forward (F) and reverse (R))
- DNA or cDNA samples, diluted to approximately 100 to 125ng/uL
- a positive control DNA/cDNA sample (a DNA sample that you know to contain the sequence that your
primers target)
- at least one negative control DNA/cDNA sample (a DNA sample that you know does NOT contain the
sequence that your primers target). Most laboratories use water but you may also want to use a second
negative control that contains non-target sequences.
- PCR Master Mix (we buy ours premixed; cat#) but you can make your own by adding dNTPs, Buffer,
MgCl2, and Taq polymerase
- ultrapure water
Lab equipment needed:
- Thermocycler
- Thermocycler-specific tubes (individual or strips) or plate
- Micropipette with sterile tips
- a multi-channel pipette is also useful for some parts of the protocol if you are using a plate or
tubes that are connected
1. Create Master Mix, if making your own. Be sure to make enough for 110% of your samples plus
controls to account for pipetting error.
2. Dilute your primers to the appropriate concentration.
3. Dilute your DNA to approximately 100-125ng/uL.
4. When using the pre-mixed Master Mix we add 9.5uL ultrapure water to each pre-labeled tube or
wells of your plate. Then mix in 1uL of each of the primers which had previously been diluted
to 10uM, 12.5uL of the Master Mix and lastly, 1uL of diluted DNA and you controls, one per
well. Running your samples in duplicate is not a bad idea.
a. If you are making your own master mix, your PCR conditions will need to be optimized.
I suggest consulting:
i. McPherson, M.J. 2000. PCR. London : BIOS Scientific Publishers.
ii. Roux K.H. 1995. Optimization and troubleshooting in PCR. PCR Methods and
Applications. 4: 185-194.
5. Run your PCR on your thermocycler. This will need to optimized to your PCR reaction and
thermocycler.
a. PCR cycling usually starts with an initial denaturation for 1 to 5 minutes, followed by 20
to 50 cycles of denaturation usually at 94C (92-96C) for 30 seconds, primer annealing
which usually lasts 30 to 60 seconds (see below for temperature information), and
elongation/extension at 72C (which is at the temperature at which Taq performs
optimally), and is terminated by a longer extension step, again at 72C, for 5 to 10
minutes.
i. To find the appropriate annealing temperature to begin optimization, it is often
best to start by determining the melting temperature of your primers by using the
equation: T m = (G + C)4 + (A + T)2. The temperature usually falls within the 40
and 70C range. Then test the temperatures ~3-5C above and below that
temperature.
b. Again, I suggest consulting the following for more information on optimization:
i. McPherson, M.J. 2000. PCR. London : BIOS Scientific Publishers.
ii. Roux K.H. 1995. Optimization and troubleshooting in PCR. PCR Methods and
Applications. 4: 185-194.
**Contamination is the bane of the PCR user. Be very careful to avoid contamination by:
1. Using sterile pipette tips and tubes. Positive displacement pipettors
prevent aerosols and reduce the chance of sample carryover. Pipette tips
with filters are also a good idea.
2. Sterilizing the area you are going to be preparing PCR reactions in prior to
use. This can be done with 10% bleach (but be careful not to get it on
your clothes) or special cleaning products such as DNAway (cat#).
3. Wearing gloves and changing them frequently.
4. Preparing your PCR reactions away from the thermocycler. We do our
reactions in a separate room.
5. Add your DNA to reaction tubes last.
DNA ELECTROPHORESIS
Materials:
- Agarose I (cat#61-412-RR)
- 1x TAE
- ethidium bromide (EtBr; cat#)
- loading dye (cat#)
- ladder and sample DNA (such as from a PCR reaction)
Lab equipment needed:
- scale
- microwave
- microwave-safe flask (e.g. glass)
- gel tray, combs, and gel chamber (e.g. fisher cat#S98616)
- micropipette and sterile tips
- UV light box and associated PPE
1. Prepare the gel
a. We make 0.7% gels in 100mL capacity gel trays.
b. To do this, mix 0.7g Agarose I with 100mL 1x TAE.
c. Microwave this mixture on half power for 30 second
intervals until the agarose is dissolved. **Be careful
as the mixture will be very hot!!
d. Allow the mixture to cool until it can be handled.
e. In the meantime, prepare the gel tray by taping the
two open ends (see pictures to right).
f. Then add 10uL of Ethidium bromide to the mixture.
EtBr in addition to the loading dye allows the
visualization of DNA with the use of a UV light box.
g. Pour the mixture into the gel tray.
h. Add a comb to the tray to create the wells where
DNA will eventually be placed.
i. Allow it to solidify (approximately 30 minutes for a
100mL gel tray, depending on how cool the mixture
was before it was poured).
j. Remove the tape from the edges and the comb from the gel. Then load the gel into the
chamber by setting it directly onto the chamber’s platform while keeping the gel in the
gel tray (**caution: gel is very slippery).
k. Fill the chamber with 1x TAE until the gel is completely covered so that current can flow
throughout.
2. Prepare and load the DNA
a. For every 5uL of a DNA sample, add 1uL of loading dye. This can be done in
microcentrifuge tubes but a cheaper, simpler method is to mix the DNA and dye on a
strip of parafilm.
b. Load at least one well per line with DNA ladder by loading 5-6uL of the dye into a
micropipette with a sterile tip and putting the pipette tip into the bottom of a well created
by the comb. Be careful not to push the pipette into the gel. We generally add the dye to
one of the middle wells to aid in visual inspection but other labs put them on both ends of
the line. The ladder will allow you to determine the size of your unknown sample bands.
c. Load the DNA- dye mixtures into individual wells.
3. Run the gel
a. We generally run our gels for 45 minutes at a constant 120 volts. Make sure the negative
voltage is closest to the where your DNA is loaded so that the DNA has room to move
towards the positive charge.