Radioactive probes by PCR.

dsfsd
Mon Mar 20 08:06:27 EST 1995


I should like to thank everybody who answered my question about generation
of radioactive probes for Southern by PCR.The following protocol (taken
from Schowalter & Sommer (1989) Anal.Biochem.177,90-94) gives really good
short (94bp) hybridization probes:
1) 94bp template DNA was purified from agarose gel.
2) 20u PCR reaction mix contained:
10ng (1u) DNA template
1.6u of 25mM MgCl2
2u of 2mM dA,T,G mix
5u 50uCi 32P labelled dCTP
1.6u of each specific PCR primer (final concentration 0.4uM)
2u of 10*PCR buffer mix
1u of Taq polymerase

30 cycles were performed: annealing 2min at 55C,elongation 3min at
72C,denaturation 20sec at 96C.
3) The reaction was cleaned by Sephadex column.
The incorporation was about 60% and this short probe worked very good in
screening of cDNA library.



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