I would suggest overlapping PCR. Basically you need two pairs of primers - outer primers that are positioned over unique RE sites preferably close to your mutation and two primers introducing your mutation - first you make 2 PCR reactions -(left and right parts) , and then you combine the two with only outer primers for 2nd PCR to join the two. At the end digest with RE and clone. If you want to do less screening you might want to include silent mutant RE (close to your mutation) site for easier identification of recombinants. Hope this helps. Peter
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