a question on PCR of bacterial colonies
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
> : : 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
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
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
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
e-mail kraev at bc.biol.ethz.ch
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