Labelling PCR products for detection
kirov at medfac.acad.bg
Thu Nov 6 03:09:35 EST 1997
Pawel Golik wrote:
> Hello All,
> I want to label my PCR products (microsatellite VNTR analysis) for
> subsequent detection in polyacrylamide gels (the sequencing gel type - I
> need to discriminate alleles differing by as little as 1-2 bp). I was
> advised to use radioactivity (I considered silver staining as well).
> I have seen one protocol, using in a 11 ul PCR reaction 0,5 uCi of
> alpha 32P dCTP and a mix of 200 uM dATP, dTTP, dGTP 10 uM dCTP.
> Will this be enough? Will the decreased dCTP concentration cause
> problems in amplification (like more polymerase stuttering etc.).
> I don't want to end-label primers - I'd have to buy gamma 32P dATP just
> for that, while alpha 32 P dCTP is routinely used in our lab. Using like
> 50 uCi per reaction, as some protocols suggest is also out of question.
> Anyone with advice?
> Pawel Golik
> Deartment of Genetics
> Warsaw University
I don't think you need radioactivity, but if you want sunburns, put some
cold dCTP (for example 50 um cold dCTP final concentration). This will
Medical Faculty, Sofia
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