Synthetizing probes with Taq

Zal Suldan z-suldan at SKI.MSKCC.ORG
Fri Nov 4 08:06:21 EST 1994


>Guy MORDRET
>mordret at sb-roscoff.fr

>Does anyone has a protocol to synthetize labelled probes using Taq Polymerase
 >and a 32P dNTP ? Why the yield of incorporation of a labelled dNTP is lower
 >using Taq Polymerase and two specific oligos rather than using the klenow
 >and the random priming method ?




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
rest.

Hope it works for you!

Zal

_________________________________________________________________
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 ski.mskcc.org    or   ZSuldan at Stud.Med.Cornell.edu





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