PCR Screening of transformants?
Koen De Smet
k.desmet at nospam.ic.ac.uk
Tue Dec 16 04:06:29 EST 1997
Dom Spinella wrote:
> > Hi folks --
> > I desperately want to get PCR screening of transformants going, and
> > I have gotten it to work inconsistently in the past. The problem is
> > that most of the time it did not work. Is there anyone out there who
> > has figured out most of the sensitive steps (eg how much bug colony to
> > use, how to harvest the bugs, whether to pre-explode the bugs, etc) for
> > this sometimes wonderful, sometimes useless technique.
> > Thanks,
> > Harry
> Strange thing about colony PCR -- people either swear by it or swear at
> it. I suspect that the variability of the results is a function of the
> strain of bugs, the plasmid vector, or the PCR primers rather than the
> PCR protocol per se. So for all you folks who get colony PCR to work
> "every time" (as well as you folks for whom it often fails) what host
> strain, plasmid, primers and amplification conditions are you using?
> Maybe there's enough data out there among all of us who have tried this
> technique to draw some conclusions and generate a "virtual" methods
> paper without any one of us having to do all the experiments!
> I can tell you that in my experience, colony PCR works well with
> pBluescript or pUC plasmids in DH5-alpha cells using M13 forward and
> reverse primers, but works poorly or not at all with pZero in STABL2
> cells and the same primers. In both cases, we just pick colonies with a
> sterile pipet tip and add the bacteria directly to a standard 50 ul PCR
> mix. We do attempt to lyse the bugs with an extra 2 minute heat
> denaturation at 95 degrees prior to the first amplification cycle.
> If you have similar data that you'd like to share, cc my e-mail address
> in a reply, I will tabulate and post the results.
> -- Dom Spinella
I wrote this before in this newsgroup, but I may just refresh your
memory: I have done PCR on genomic DNA from E.coli, spirochetes and
mycobacteria, simply by suspending a pellet of a small amount of bugs
(say from 50-100 ul culture) in 100 ul chloroform, vortexing, adding 100
ul water, vortexing and spinning. 2-5 ul is the nused in a 50 ul PCR.
I haven't done it to screen for transformants, but a colleague of mine
has and says it is better than the boiling method. The chlorophorm lyses
the cells, and maybe also removes some of the inhibitors from the cells?
Koen De Smet
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Imperial College School of Medicine at St Mary's
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