DNA Extraction for PCR.
Leonard N. Bloksberg
bloksber at pilot.msu.edu
Mon Oct 18 11:32:00 EST 1993
In Article <11OCT93.13545881 at msdisk.wustl.edu> "wetsel_r at msdisk.wustl.edu" says:
> In a previous article, Yves Brun <ybrun at bio.indiana.edu> wrote:
> >>Greetings Netters:
> >>I have a quick question for you: What reference do you use, or protocol
> >>do you use for the extraction of genomic DNA for use in PCR? I'm not
> >>looking for something that would produce DNA of such quality for genomic
> >>Southerns, we have those protocols and they take 2.5 days! I'm
> >>looking for something quick, easy, and reproducible where I can spend
> >>time thinking about the PCR reaction than the DNA extraction!
> > I don't know what your source of DNA is but this is what I use for
> >bacterial genomic DNA (Caulobacter crescentus, a Gram-negative). It is
> >fast and gives very high quality DNA.
> Yves: Thanks for the reply... my mistake. I'm looking for
> a protocol for eukaryotic tissue, specifically tissue culture
> I'm using it as a check to see if stably transfected cDNAs
> within an expression vector are present in some form or another.
> So my goal is to take a small sample of the stable transfectant, shortly
> after removal of G418, and test them to see if the contruct is present
> prior to expanding, sublcloning, and using much labor and $$$.
> I have one transfectant which seemingly has kicked out the cDNA but
> retained G418 resistance as determined by immunoprecipitation and southern
> analysis. So basically, I'd like to check my other transfectants via PCR.
> Thanks again,
> haviland at kids.wustl.edu
When you say eukaryotic cells, do you mean plant or animal? The DNA
extraction procedures differ significantly.
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