grimwade at curagen.com
Mon Sep 18 14:49:36 EST 1995
In Article <43ah37$m3c at mail.llu.edu>, Jeff Sale <jSale at ccmail.llu.edu> wrote:
>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.
I used to have problems with this, too. The trick is to spike the hot
nucleotide with a little unlabelled, so that there is enough around to allow
efficient amplification. Empirically, I found that 1ul 5mM d(AGT)TP, 5 ul
32PdCTP, 1 ul 50uM dCTP gave good PCR labelling, and lots of product. If I
tried it without the cold spike, I'd get <5% the incorporation, and lousy
hybridization. Hope this helps,
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