Labelling PCR products for detection

Pawel Golik pawel at ibbrain.ibb.waw.pl
Wed Nov 5 11:22:29 EST 1997


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



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