dna at mail.vt.edu
Wed Apr 5 08:59:48 EST 1995
Subject: multiple bands
From: Ann M. Yezerski, ayezersk at moose.uvm.edu
Date: Tue, 4 Apr 1995 14:43:25 GMT
In article <1995Apr4.144325.4283 at emba.uvm.edu> Ann M. Yezerski,
ayezersk at moose.uvm.edu writes:
>I need any and all suggestions for getting rid of multiple banding
>patterns on a PCR. Many I'm sure I've tried, but I need some fresh
>ideas. I using Kocher et al.'s universal 12S primers on lizard
>extracts. My best results have come from a ramped 45 degree anneal for
>one minute, and all other periods also for one minute. Generally I use
>microliter of MgCl2 per 50 microliter reaction. My results
>produce one, two, or all of the following sized bands:378 (the right
>approx. 150, and approx 50!
>If you any suggestions, write to me at ayezersk at moose.uvm.edu
I've had some similar problems with PCR and a technique which worked for
me was in biotechniques about a year ago called touchdown PCR. This is
where you ramp the annealing temperature down over the first few cycles
to eliminate any nonspecific binding.
I'm sorry I don't have the ref. (also for the authors for credit's sake),
but it has worked well for a couple of labs here at VA Tech.
Also, try varying the MgCl from .5 to as high as 6 or 7 mM.
Good Luck. And may the Force be with you!
John R. McQuiston
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