Colony screening with PCR

BOIS, DANUTA H dhb at gnv.ifas.ufl.edu
Fri Aug 18 10:23:01 EST 1995


I've been using PCR to screen colonies for inserts of interest.  I prepare
a miniprep DNA from bacteria using the alkaline lysis method and use that
in the PCR reaction.  Often it works great, I get a nice bright band.
However, often enough it doesn't work at all, even when I know the clone is
in there somewhere, because I got a strong positive when I tested a pool
of colonies, and then after doing them individually I may get nothing.
If at that point I do another miniprep, I may get a positive response.
I feel, therefore, this has something to do with the DNA minipreps.  I don't
mind redoing them sometime, if I already know I've got a positive in there.
I am more concerned about missing some positives when I do a primary screeen
with 50 or a 100 colonies pooled together.  At that point I don't know there
is nothing there or the PCR reaction just didn't pick it up.  I've tried
doing PCR with colonies put in directly in the PCR reaction (picked with 
a toothpick or with a pipette tip) and found that method to be even less
reliable.  Would anyone have any idea as to what might be inhibiting the PCR
reaction?  I would appreciate any help you might give me.
Thanks,

Danuta 



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