making PCR probes

lader at lader at
Fri Sep 26 18:02:38 EST 1997

In article <timt-2309972220520001 at>,
  timt at (Tim Tian) wrote:
> What is the best method to make PCR probes for Northern:
>     end labeling with T4 polynucleotide kinase,
>  or throwing in hot dATP to do more pcr cycles?
> In the latter case, how effecient will the labeling be since overwhelming
> majority of dATP molecules in the PCR rxn will be cold ones?

Neither of these approaches will work. Both will yield probes of too low
specific activity. Either random prime it, or try assymetric PCR for a
few cycles with only hot dATP (no cold) or try primer extension with a
gene specific primer and exo minus klenow. These approaches may work in
your hands, maybe not.

Alternatively, add a phage promoter by making a chimeric pcr primer or use
Ambions lign'scribe kit and make a hot antisense RNA probe.

lader at

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