a question on PCR of bacterial colonies

Troels Wind wind at biobase.dk
Mon Jan 6 05:18:52 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


: Ed Taboada
: Molecular Genetics
: University of Ottawa
: Ottawa, Canada

I've never had false positives myself, but that doesnt prove anything
ofcourse...

One suggestion: Do a negative control using a colony carrying your plasmid
WITHOUT the insert. If the 'wrong' product is not present here, well, your
cloning has f... up :)

Troels Wind
Aarhus University
Aarhus, Denmark




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