Dr. C.P. Healy
chealy at hgmp.mrc.ac.uk
Fri Oct 21 09:56:24 EST 1994
hanks to everyone who replied to my posting about isolating 5' regions of
cDNAs using inverse PCR. Here thay are!
From: PAIKS at gunet.georgetown.edu
To: chealy at hgmp.mrc.ac.uk
Subject: inverse PCR
hi chris, rewgarding your question about inverse
PCR, I tried it and worked without cutting the
circularized DNA. The main trick is when you
circularize the DNA, you have to do it in a
condition to favor intramolecular ligation instead
of intermolecular, thus you have to ligate in a
very dilute solution.
In may case, I had much better luck with ligation
mediated PCR using the kit from Takara (Takara
single site PCR kit). The kit provides six
different adoptors for the different enzymes and
use nested priming. You could easily use vector
fragments to do it but the kit is cheap enough to
justify not making the reagents myself. You could
also try Pan Handle PCR if you want long 5'
fragment described in the Biotechniques last year.
i hope this is helpful.
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