PCR probes (32 P) Q&A

Marianna Max drmax at casbah.acns.nwu.edu
Sun Apr 17 19:16:31 EST 1994


In article <jspaffor.1116840785D at NEWS.SRV.UALBERTA.CA>,
J. David Spafford <jspaffor at gpu.srv.ualberta.ca> wrote:
>In Article <1994Apr13.125331.5635 at leeds.ac.uk>, futers at bpxtal.leeds.ac.uk 
>wrote:
>>Hi netters,
>>
>>Has anyone performed a PCR reaction in the presence of a 32P dNTP and
>>then used the product to probe a Southern blot?
>>If there is any interest I will post a summary.
>>
>>                       Many thanks,
>>                         Simon    (futers at biovax.leeds.ac.uk)
>
>I have used the random priming method from BRL to screen libraries and
>Southern Blots, but I prefer hot PCR.
>
>I stopped using random priming because it doesn't work well with the small
>sized probe I am using.  50% of random primer labelling products are <= 50%
>the size of the original template.  With small pieces (<200 bp), the
>template needs to be ligated together to create a larger-sized priming area.
>
>With hot PCR probes and degenerate primers, I have been able to compare
>Southern Blots using PCR probes from cloned templates vs PCR probe from
>genomic DNA template.

Someone mentioned that one advantage to PCR probes was that one didn't have
to purify the insert away from vector. For most uses this works but if you
are screening a library or a southern that contains any of your vector
sequence be aware that your entire plasmid will be linearly amplified to
some extent and in some cases enough to cause background problems.

Max




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