a question on PCR of bacterial colonies

Jeremy Marcus marcusj at helix.mgh.harvard.edu
Mon Jan 6 12:36:15 EST 1997


: parakeet (s535290 at aix1.uottawa.ca) wrote:
: : 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

When using PCR to screen bacterial colonies, I always use 5% DMSO in the
PCR reaction; this presumably increases specificity, and it may solve your
problem.

Jeremy



More information about the Methods mailing list