deletion of large fregments - (Oct/16/2012 )

hi all!

I am using the quick-change method for site directed mutagenesis. so far, i was able to create single-base or codon change with no problem. now i need to delete a whole section of 20AA (60BP)

could this be preformed using the regular procedure? do i need special consideration when designing the primers?

thanks!

Quikchange won't work for that sort of length deletion as far as I know. You might have to do something like tailed PCR where you have two sets of primers, one set on either side of the mutation, with one of the primers in each set being tailed with a tail that is complementary to the tail on a primer in the other set. Amplify the two reactions separately, anneal the tails and then amplify the whole resulting product (you may need to go to nested PCR for this step), then clone.

thank you for your comment.

Your gene of interest is cloned into a plasmid right? I think you can try inverse PCR.