ANDREI.POPOV at bbsrc.ac.uk
Fri Sep 15 12:23:21 EST 1995
>I'm trying to do hot PCR to generate a radiolabeled probe with high
>specific activity, at least higher what end-labeling would give me.
>Others in my lab have tryed but their effort yielded probes were not
>nearly as hot as probes generated by random priming.
>What can I do? I would like to avoid random priming since that could
>generate probes of various lengths. Could I just decrease the [dNTP]
>and allow a longer extension time?
I use for labelling a simple protocol which I saw in one
of the textbooks on PCR (though it has nothing to do with PCR, apart
from the starting template, which is usually, but not necessary a PCR
Rp.: DNA template ( in my case 274 bp) 20 ng
dATP, dTTP, dGTP 20 mkM end conc
dCTP (32P) 10mCi/ml 10-15 mkl
one primer 10 mkM end conc
Cycling: 95C- 2 sec
ann T- 1 min
72C - 3 min
after 20 cycles incorporation is approx. 50% which is enough for
genomic Southern blots.
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