making PCR probes

lader at ambion.com lader at ambion.com
Fri Sep 26 18:02:38 EST 1997


In article <timt-2309972220520001 at wenzi.hip.berkeley.edu>,
  timt at itsa.ucsf.edu (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 ambion.com

-------------------==== Posted via Deja News ====-----------------------
      http://www.dejanews.com/     Search, Read, Post to Usenet



More information about the Methods mailing list