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.

Paste your primer sequence below (5' to 3'):
(Only considers A, T, G and C, and is case-insensitive)

Number of mismatched bases (if there are any):

Always check your final primer sequences. Send comments and suggestions to dekim@u.washington.edu. Created by David Kim.

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http://depts.washington.edu/bakerpg