For the amino acid sequence place either spaces between all codons or
1, 2 or 3 before the sequence to start translating at the 1st, 2nd or
3rd base, respectively. According to the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) protocol, the
mutation should be in the middle of the primer,
and the primer should have a minimum GC
content of 40%, should terminate in one or more C or
G bases, should be between 25 and 45 bases in length,
and have a melting temperature close to or above 78
degrees C. The equation used for the melting temperature is: Tm = 81.5 + 0.41(%GC) - 675/N - %
mismatch, where N = total number of bases.
Always check your final primer sequences. Send comments and
suggestions to dekim@u.washington.edu. Created by David
Kim.
http://depts.washington.edu/bakerpg