labelling with PCR

Zal Suldan z-suldan at SKI.MSKCC.ORG
Wed Dec 14 19:39:37 EST 1994

I sent this to the group a while ago... but I'll send it again in response
to a recent query.

We END-label one (for footprinting!!) (or two) of the primers with PNK
using 5-10 uL of  gamma-p32-rATP (50-100uCi) to approx 25pmol of the primer
(do this in 10-20uL). If the primer is large enough (>30b), you can send
this through a p10 spin column (clontech, pharmacia, boeringer, etc),
otherwise EtOH ppt overnight, or even use straight. Add all 25pmol to the
pcr reaction and PCR away. Remember to do the positive and negative
controls like you usually do. Some of use sequencing Taq in the rxn
(eliminates 5'->3' exo and therefore won't de-label your probe), some of us
use regular Taq. Check aliquot on an agarose gel via UV and/or
autoradiograph (should need a VERY short exposure), then gel purify the

Hope it works for you!


Zal Suldan
Tri-Institutional MD/PhD Program - Department of Cell Biology and Genetics
Memorial Sloan Kettering Cancer Center / Cornell University Medical College
Replies to: Z-Suldan at    or   ZSuldan at

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