One product has been giving me serious problems. I've designed primers of varying lengths (20-25 bp) for amplicons that range in size from 200-800 bp. I've tried modifying primer concentration (200nM - 1000 nM)... Nested PCR reactions, Mg2+ optimization, touchdown, reverse primer alone for 10 cycles, HotStart, etc. However, for the life of me, all I get is a smear.
Are there regions in the genome which are just not amplifiable via bisulfite? Any other tips?
Edited by unknownphd, 28 April 2004 - 03:02 PM.