I'm trying to use shotgun alanine scanning to test the importance of 7 key residues in a protein. Can anyone give me advice on how best to achieve that many simultaneous, consecutive mutations?
I've made myself a tool to find minimally degenerate codons, but am not sure how I'll actually do the mutagenesis. Would it be foolish to try to design one long PCR primer (following the Megaprimer mutagenesis method) that incorporates all 21 bases? I'm assuming that I'll need at least 20 BP on either side to anchor the primer down. This would mean using a 65mer.
When I analyzed this using the IDT oligo analyzer, it was throwing up a heap of secondary structures and self dimerization. Can anyone give me advice on tools to use when designing such long primers or tell me if there's a better way to do it?
Edited by jeremyn, 26 July 2010 - 02:02 AM.