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colony PCR for E.coli genomic sequences

Lori lcp at vetmed.wsu.edu
Mon Dec 8 17:06:58 EST 1997


Richard Koepsel wrote:
> 
> Netters,
> Does anyone have experience with detecting E.coli genomic sequences
> using colony PCR?  We find ourselves in the position of having to screen
> for a deletion (which we are trying to construct) that has no selectible
> phenotype.  We would like to avoid colony screens if possible.  We can
> amplify the target from purified chromosomal DNA and we can do colony
> PCR of the gene cloned in a pUC plasmid.  Any suggestions?
> Thanks,
> Rick Koepsel

Rick,
In my previous job, I analyzed thousands of enterotoxigenic E.coli
isolates for toxin genes (on the chromosome) by colony hybridization. 
Using a 96 well plate format, I was able to screen 8 plates (more than
700 isolates) for two different genes, with a total of about 20 hours of
lab time over 4 days.  (I used the Genius kit, with chemiluminescent
detection.)  For our screening purposes this was much more efficient. 
HOWEVER, I also routinely do PCR from these bugs with a super easy
prep...just suspend a colony in 300 microliters of water, boil it for 20
min. and use 5 microliters in a 50 microliter PCR reaction. (This is a
protocol I got from the CDC and it always works...I've used it to
amplify Salmonella genomic sequences, too.)
Good Luck!
Lori



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