The "megaprimer" method of PCR

[the End]

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.

Jim
J. Graham PhD
Biology Department 
Washington University of St. Louis