Labelling PCR products for detection
pawel at ibbrain.ibb.waw.pl
Wed Nov 5 11:22:29 EST 1997
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?
Deartment of Genetics
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