Want PCR to incorporate 32P label
Manuel Maria SIMON
Simon at mail.boku.ac.at
Mon Mar 13 10:18:53 EST 1995
smori at nmsu.edu (Shahram Mori) wrote:
> PAUL JONES (jone3 at essex.ac.uk) wrote:
> Has anyone had success with incorporation of 32P dNTP into an extending strand
> in PCR for northern/ southern hybridisation? I would have thought is would
> higher signal / noise ratio than probes produced by random hexamer method
> and a method of choice for probes <200 bp.
> Hope there is opinion out there.
I did PCR labelling successfully. I amplified some 0,5 ng
(or less) plasmid in a normal buffer system (e.g.1,5 mM Mg++).
Instead of the 100 microM dNTP I used 5 in each except the 32P-dCTP
(3000 Ci/mmol from NEB), which is usually 50 microCi.
Then a 20 cycle PCR was done in conditions where I knew that the
fragment was properly amplified, e.g. as a single and expected band.
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