a question on PCR of bacterial colonies

Alexander Kraev kraev at bc.biol.ethz.ch
Tue Jan 7 05:43:56 EST 1997


In article <marcusj-0601971236150001 at path-myerson.mgh.harvard.edu>,
marcusj at helix.mgh.harvard.edu (Jeremy Marcus) wrote:

> : parakeet (s535290 at aix1.uottawa.ca) wrote:
> : : Hey all,
> : 
> : : I always check inserts of my plasmid clones by PCR before I go about
<SNIP<
> : : 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

Hi there,
there are definite cases when colony PCR is screwed up by an insert's sequence.
One clear case is GC-richness. In such cases no product  is obtained from
a positive clone,
in contrast to clear small bands from"empty" clones.  Adding 5% DMSO only
partially solves
this problem, as there are still cases where this is not enough to lower
the melting temp of the
insert's duplex.  One can go up to 12.5% and double the amount of
polymerase, however,
even this may not always help.
In cases where the clone is also a low copy number ( also determined by an
insert rather than
the original vector), colony PCR often gives a double band,  of which the
larger one is the correct one,
and a smaller one is somehow generated from the Lac sequence itself,
perhaps from a neighbouring
normal copy number empty clone.
Combine the two, and you get a clone that is not "colony-PCRarable"! Could
that be your case?

Just my two cents :-)

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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