Hot PCR

[Brian Grimwade]

In Article <43ah37$m3c at mail.llu.edu>, Jeff Sale <jSale at ccmail.llu.edu> wrote:
>Hi,
>
>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.
>
(snip)

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,

BGG