Richard Koepsel wrote:
> 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?
> Rick Koepsel
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.)