multiple bands

John McQuiston 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
1 
>microliter of MgCl2 per 50 microliter reaction.  My results 
>produce one, two, or all of the following sized bands:378 (the right
one), 
>approx. 150, and approx 50!  
>
>If you any suggestions, write to me at ayezersk at moose.uvm.edu
>
>Thanks.
>
Hi Ann,

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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