Hot PCR

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Fri Sep 15 12:23:21 EST 1995


>I'm trying to do hot PCR to generate a radiolabeled probe with high
>specific activity, at least higher what end-labeling would give me.
>Others in my lab have tryed but their effort yielded probes were not
>nearly as hot as probes generated by random priming.
>                                     
>deleted......
>
>
>What can I do?  I would like to avoid random priming since that could
>generate probes of various lengths.  Could I just decrease the [dNTP]
>and allow a longer extension time?  

Hello Tony,
I use for labelling a simple protocol which I saw in one 
of the textbooks on PCR (though it has nothing to do with PCR, apart
from the starting template, which is usually, but not necessary a PCR
product).
Rp.:  DNA template ( in my case 274 bp)        20 ng
      dATP, dTTP, dGTP                          20 mkM end conc
      dCTP (32P) 10mCi/ml                      10-15 mkl
       one primer                              10 mkM end conc
       Taq buffer
      Taq pol


Cycling: 95C-   2 sec
         ann T- 1 min
        72C   -  3 min
---------------------
after 20 cycles incorporation is approx. 50% which is enough for
genomic Southern blots.

 
Good luck

Andrei Popov




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