general question about PCR
Bernard Murray, PhD
spam at 127.0.0.1
Fri Nov 12 22:13:00 EST 1999
In article <80iio8$onb$1 at nnrp1.deja.com>, jennnn1970 at my-deja.com wrote:
> Hi all... this is a very basic question about PCR.
> I understand the process of collecting tail DNA, making of primers, the
> thermalcycles and such. I just don't understand how you'd get a bunch
> of fragments to run on a gel at the end of the PCR?
> Do you digest the genomic dna first? Do you digest the PCR'd DNA at the
> end? Do the primers only extend so far?
> I don't understand why the PCR reaction wouldn't copy the WHOLE piece
> of DNA.
> Confused and can't find any answers in textbooks or the web,
Not sure what you are asking but it sounds as if you are screening
transgenic or knockout mice etc. by PCR of tail DNA. When I've
screened Tg animals I've just picked a pair of primers that
amplified a piece of DNA specific to the transgene (eg. upstream
in the promoter, downstream in the cDNA) to give a fragment suitable
for routine electrophoresis (eg. 200 - 1000 bp).
If you are trying to distinguish small differences eg. between
related mouse strains, you may need to cut the PCR product with an
enzyme that distinguishes between the two sequences, or there may
be a size difference between the two.
You don't have to do anything special with the tail DNA before
PCR. I use overnight digestion with proteinase K and then spool
out the DNA after treatment with 2-propanol. Rinse with 70%
ethanol "on the spool" and then dissolve in TE. Fine for PCR
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
More information about the Methods