primer design for PCR cloning - (Sep/03/2007 )
I am designing primer pair for PCR cloning. I just want to ask what would be more feasible, to add few A,s or mix of different nucleotides after the restriction site and how many of them should be there??? Please help....
I would prefer a mix for the guard sequence, leaning a little more heavily on GC.. perferably starting the primer with either 5'-GC or 5'-CG (GC camp). The 5' end of the primer should bind stronger then the 3' end allowing the 3' to search of the most ideal basepair complementation.
The number of nucleotide added depends on the restriction site. For most restriction enzymes 6bp of skirting is more then enough. However for some restriction sites like NotI and NdeI, 8bp is the minimum number of skirting bp required for the restriction site to be cut at any efficiency. NEB technical guide is a good place to look up for this infomation.