a question on PCR of bacterial colonies

parakeet s535290 at aix1.uottawa.ca
Fri Dec 27 20:53:04 EST 1996


Hey all,

I always check inserts of my plasmid clones by PCR before I go about
doing preps. I've never had a problem, and I'm not convinced I have one
now either. I have been trying to clone this one piece of DNA and keep
screening white colonies by PCR, only to find products which are too small
to be what I'm looking for. Since this little endeavour is of outmost
important, the thought that somehow some of these white colonies actually
do carry an appropriate insert, but that something could go wrong in the
PCR has been gnawing at the back of my brain. 

So the question to you who use PCR screening of bacterial colonies is if 
there is any possibility for false negatives. The piece I'm trying to
clone is 1.7 Kb and I do use primers that are about 150 bp from the
cloning sites on either side of the MCS, so the expected product would be
around 2 Kb. It may very well be that my cloning just isn't working (a
definite possibility ! :)) but if any of you has ever encountered false
negatives that actually carried the inserts of interest, I'd like to hear
from you.

thanks for any help.

Ed


Ed Taboada
Molecular Genetics
University of Ottawa
Ottawa, Canada





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