T4PNK 5'-P of primers: pre-PCR cleanup?

Steve Eklund seklund at MolBio.unmc.edu
Fri Jan 7 18:22:04 EST 1994


I need to 5'-phosphorylate some primers for use in the PCR half
of RT-PCR with the P-E GeneAmp kit, so the amplified product can
be blunt-ligated into a phosphatase-treated plasmid site.

Here's my question: After the T4 PNK phosphorylation, how much
cleanup is necessary before adding the primers to the PCR
digest?  Is any cleanup necessary?

Maniatis and Current Protocols discuss methods for purifying
radiolabeled primers and probes, but the oligo concentration in
these reactions is much lower.  One colleague told me that she
adds the T4 PNK digest directly to the PCR digest.  Any
opinions or words of wisdom?







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