RAPDs and PCR

Gregory Buzard buzardg at MAIL.NCIFCRF.GOV
Thu Feb 22 18:34:33 EST 1996


We have had experience PCR amplifying bands punched out of gels and the
accompanying artifacts. Two main types: 1) Surface contamination from buffer
and staining trays (if non-isotopic), 2) the "hay falling off the wagon on a
bumpy road affect".
1) Prove to yourself, as we did, that this is a problem. Punch out any
non-lane region of the gel and amplify as usual. You will get most of your
smaller products that are causing you problems. For grins, do the same thing
for a ul of running buffer. The cure: Rinse your gel boxes and staining
dishes with 5% v/v bleach for 5-10 min., and then rinse extensively with
water before running gels which you might want to punch out. Handle with
some, but not fanatical, care while staining or drying and exposing. The
small stuff is diffusing out as you stain, so you must work relatively
quickly to minimize the surface contamination that will occur over your
band. Just before punching out wash 2-3 times with a good volume of clean
water or buffer to remove as much surface material as possible, and take as
little as possible of the band (we use a 200 ul tip to just punch the gel).

2) Again, prove this to yourself. Take plugs at 3 mm spaces above and below
what you consider to be a strong clean band of moderate length (200-500 bp).
Amplify for the band a minimal number of cycles. Plugs from below the
desired band will not make a product of the strong band size, and all the
plugs above will, to a surprising distance. We think this is from two
causes: A) some DNA molecules become temporarily or permanently trapped in
the matrix as the main band passes, and B) molecules that have only
partially completed extension of one of the two strands, or which are
unpaired as in asymmetric PCR, are retarded in migration in a manner similar
to an SSCP gel. The full length strand is acting as your template.

The small sized bands and blurs below your band of interest are leaving
behind nasty surprises for you! And since diffusions from the gel plug
favors smaller sized molecules, and PCR prefers to make the smallest
products possible, given a choice, you get lots of undesired small bands on
re-PCR.

The cure: Size fractionate your product on an agarose gel prior to running
on the RAPD gel, Don't overload the DNA, Let your sample sit at room
temperature for 30 min. then give it a 5 min shot at 72 degrees to polish of
any partial extensions and asymmetric amplified ssDNAs, Give your elution
much more time, and your extension steps in the re-PCR more time, to favor
the longer molecules you hope to amplify.




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