The "megaprimer" method of PCR
lruble at bronze.ucs.indiana.edu
Tue Jan 30 10:22:53 EST 1996
I have been able to get the second stage to work when the first
product was realtively short (300 bp) but not when if was larger.
I did try many of the modifications including using a high
concentration of template (~3 ug) in the second PCR.
Try gel purifying the first round PCR product (megaprimer) on
low melting agarose and using it directly as a gel slice
in the second round with "lots" of clean template DNA. You
may have to change the duration of cycle steps as well.
I believe I gave up in one case with a megaprimer of about 600 bp.
J. Graham PhD
Washington University of St. Louis
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