PCR probes by incorporation of 32P ?

Joseph C. Bagshaw jbagshaw at wpi.edu
Fri Mar 8 14:03:58 EST 1996


I've had good sucess with plasmids based on pBluescript, T3 and T7 
primers and the following recipe:

10 microM each dCTP, dGTP, TTP
5 ul {32P}dATP (50 microCi)
2.5 milliM MgCl2
primers at 10 microM each
cycle 30 times with annealing at 55 deg

Hot stuff!

This is based on an article in Focus two or three years ago.  I can dig 
out the reference if you need it.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.

On 1 Mar 1996, Geoffrey Neale wrote:

> 
> Hello everyone.
> 
> I am looking for a method for making 32-P labeled probes during PCR. The
> information that I specifically need is the concentration of cold 
> nucleotide in the reaction so that you generate sufficient full length 
> probes, but still retain high specificity of the isotope label.
> 
> Other considerations are the amount of starting template, the 
> concentration of primers, and the number of cycles.
> 
> Thanks for your input.
> 
> Geoff Neale
> 
> PS Could you please email me your advice since our news reader is not yet 
> operational on our system. E-mail is my only access at this time. Thanks.
> 
> geoffrey.neale at stjude.org
> 
> 



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