a question on PCR of bacterial colonies

Paul N Hengen pnh at ncifcrf.gov
Fri Jan 17 13:52:16 EST 1997


Ed wrote:

> 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.

| 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

Alexander Kraev (kraev at bc.biol.ethz.ch) wrote:

: 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?

Ed,

One other problem in your cloning of the fragment might be that the gene is
somehow toxic to the cell and parts of it are being deleted to give you the
white colonies. Sometimes you won't even get the light blues as mentioned
by someone else because of this problem. The question is whether you can PCR
the fragment using your primers from the original genome or from a larger
cloned piece of DNA. That control is important because it will tell you if
the DNA is able to be amplified by PCR. If you can, you might have the toxic
gene problem. If this is the case, you might have to clone the smaller fragment
into a genetic background with the larger DNA fragment on a compatible plasmid.
If you need more info about how the smaller fragment might be unclonable,
take the shuttle from Ottawa U. to Carleton U. and read my Ph.D. thesis in
the library :-)

--
*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - -  Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - -  *
* -  TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - -  *
* - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/  *
*******************************************************************************



More information about the Methods mailing list