David-Paul Minde via methods%40net.bio.net (by davidminde from gmail.com)
Wed Oct 7 14:49:03 EST 2009

if you can, design primers with silent mutation to check immediately for
positives by restriction analysis.
Then indeed prolongation of primers can help to get more positives. Also
extension of DpnI treatment (or different mutagenesis strategy with
exponential amplification as in NEB kit, which however requires more
expensive ultra-homogenous primers) to reduce background (also make sure you
mix properly before incubation and avoid condensation artefacts).
Phusion polymerse (or derivatives of that idea) is better for big amplicons
(works sill perfectly for mutations in a 15 kbp plasmid).

More information about the Methods mailing list