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Molecular
Biology Protocols |
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7. Agarose Gel Electrophoresis of PCR Products
1. Depending on the design of your
electrophoresis trays, either tape the ends with TimeTape
or place the tray in a gel-forming clamp.
Place a well comb in position, making sure that about 1 millimeter of
space exists between the bottom of the comb teeth and the surface of the tray,
to prevent leakage of sample out of the well.
2. For a typical mini-gel, weigh out 0.32 g
agarose directly into a 125-ml flask and add 40 ml 1X TBE buffer. (Larger gels will require proportionately
larger amounts of agarose and TBE).
Cover the flask with saran wrap. Microwave about 1 minute. Check that
all the agarose has dissolved. (The flask will be hot! Handle with folded paper
towels.) Microwave for additional 10-second intervals for
complete dissolution. Allow to cool to about 60C (hot but holdable in the hand) and pour gel into electrophoresis
tray with well comb in place. Allow to cool completely without disturbing
(about 20 minutes).
2.
PCR samples produced with RedTaq polymerase are ready
to apply to the gel. For samples
produced without RedTaq, label enough microcentrifuge tubes (0.65 ml) to accommodate your PCR
samples plus one DNA size marker. Add 1 μl
Loading Dye and 10 μl of PCR product.
3.
For the DNA size marker, use Low DNA Mass Ladder (1 μl)
+ 10 μl 1X TBE + 1 μl
Loading Dye.
4.
Remove the tape from the gel tray and place the in the electrophoresis chamber
with the sample wells toward the negative (black) electrode. Fill the gel tank
with about 300 ml of 1X TBE buffer to cover (barely) the gel surface. Gently remove the comb. Make sure that the
well “lips” are covered completely with 1X TBE.
5.
Load wells with 11 μl PCR sample (or size
standard), using a P20 micropipette.
Place the pipet tip barely in the top of the
well but be careful not to puncture the agarose at the bottom of the well. Dispense the sample into the well slowly. Be sure to record the pattern of sample
loading.
6.
Hook up electrodes (red to red, black to black) and electrophorese
samples toward the positive electrode (run to the red!) at a maximum of
100 volts. Allow marker dye to travel one-half to two-thirds the distance to
the end of the gel.
SYBR
Safe DNA Gel Stain
1.
Slide gel into a plastic tray (pipet box cover works
great) containing about 50 ml of SYBR Safe DNA Gel Stain (Molecular Probes
– Invitrogen). Allow to stain for about 30 minutes. Protect the gel and staining solution from
light by covering with aluminum foil.
Pour stain into a used stain storage bottle. Stain may be re-used several times. but
should be filtered through activated charcoal for disposal. Dispose of the
filter as hazardous waste.
2.
Cover the surface of the Safe Imager blue-light transilluminator
(Molecular Probes – Invitrogen) with saran wrap and slide the gel onto
the saran. Smooth wrinkles. To visualize the stained DNA, cover the gel with
the amber filter or wear amber viewing glasses.
Note: The light emitted from the
Safe Imager transilluminator is NOT hazardous and no
skin or eye protection is required.
Record the gel image with the digital imaging system. Alternatively, photograph the gel with
Polaroid 667 film (about 1 sec @ f8). Develop the film for 30 seconds and
remove the photograph, carefully discarding the remaining (caustic) materials.
3.
Measure the migration distance of each DNA marker band and plot mm traveled
against log10 base pairs using GraphPad
software. Calculate the sizes of the bands in your PCR products from the
graph. Here is a scan of the DNA Low
Mass Ladder after electrophoresis and staining:
4. To recover the bands for sequencing, proceed
to the Qiagen QiaQuick Gel Extraction protocol. Otherwise, wrap the gel in saran wrap for
disposal in the solid waste.