PCR probes (32 P) Q&A

[J. David Spafford]

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.

Per blot, I have used the following hot PCR probe labelling protocol:

0.2 ug - 1 ug DNA template
PCR buffer mix
1.5 mM - 3.0 mM MgCl2
Taq polymerase

0.2 ul 100 mM dGTP
0.2 ul 100 mM dATP
0.2 ul 100 mM dTTP
0.2 ul 10 mM dCTP
------------------
in 95 ul reaction

5 ul 50 uCi 32P labelled dCTP (370 MBq/ml//111TBq/mM//3000Ci/mM from Dupont NEN)

run 10-15 cycles
separate unincorporated by (for example):  isopropanol precipitation
                                           Pharmacia NICK spin columns

I haven't spent much time optimizing the protocol, but it appears to work.
I would appreciate some suggestions for improving the protocol if someone
has some.

Thanks in advance,
--------------------------------------------
J. David Spafford
Department of Zoology, University of Alberta
jspaffor at gpu.srv.ualberta.ca
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