Want PCR to incorporate 32P label

Dr. Kent L. Nastiuk nastiuk at rockvax.rockefeller.edu
Mon Mar 13 21:57:55 EST 1995


kang at msvax.mssm.edu wrote:
>
> In article <9503101947.AA03207 at seralph6.essex.ac.uk>, jone3 at essex.ac.uk (PAUL JONES) writes:
> >Has anyone had success with incorporation of 32P dNTP into an extending strand
> >in PCR for northern/ southern hybridisation?  I would have thought is would 
> >give 
> >higher signal / noise ratio than probes produced by random hexamer method 
> >and a method of choice for probes <200 bp.
> >
> >Hope there is opinion out there.
> >
> 
> 
> 	
> I tried this method without any success.
> So far for me, random priming is the best choice.
> 
> Chulho Kang

well, i wasn't going to clutter up this thread,
but unlike Kang, i routinely make probes with pcr
by including three cold dNTPs and 50 uCi hot dCTP 
(high activity).  works every time as long as you 
remember to denature the probes.  also, use only a 
couple of ng plasmid containing your insert for 
the reaction.

i've used pcr made probes for colonies, plaques,
and southerns.  its nice becuase you don't have 
to purify insert.

good luck



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