Help: PCR screen of colonies
css at med.unc.edu
Tue Sep 14 16:18:30 EST 1999
I concur with Eric. No need to muck around!
Eric Chace Olivares wrote:
> Kirk A. Gilbert, Ph.D. (kagilbert at psghs.edu) wrote:
> : Colony lift with pipet tip, transfer to replicate plate, then pipet into
> : 50 ul water. Boil, and use 2 ul in PCR reaction with gene specific
> : primers (50 pmol ea.), 0.1 ul Taq, dNTPs, PCR buffer for total volume of
> : 20 ul. 30 cycles, annealing at 55° C.
> I'd say lower your annealing temperature a by 500 degress or so. :)
> Seriously, I do this all the time, and I don't even boil first. I setup
> the reaction with everything except the Taq, touch the colony with a
> toothpick or tip, touch to a plate, then vigorously swirl it in the PCR
> reaction. Stick it in the PCR machine, 95 for 3 minutes, add the Taq and
> continue cycling. I have done this with complete reaction mixtures
> (including the Taq) and it has also worked fine. You might be a little
> low on the Taq, as I usually use 1.25U of Qiagen Taq.
> Good luck...hopefully this helps a bit,
> Eric C. Olivares
> Department of Genetics
> Stanford University School of Medicine
> olivares at stanford.edu
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