Labelling PCR products for detection

Kirov 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

Hi,
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
work.
Good Luck!
Steven Kirov
Medical Faculty, Sofia



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