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Point Mutation in DNA - Quik change mutagenesis (Apr/17/2007 )

I got a vector in which I need to do a point mutation.

I have been using from years the Quik change mutagenesis kit from Stratagene to do that and I thought I was good at it.

This time, somehow I got a plasmid (since I get colonies after transformation) with the desired mutation (confirmed by DNA sequencing) but with 1 kb less. Anybody has an idea how this could happend?

I am doing a deletion of 3 amino acids. This might be it.

I tried ordering new primers and this time I do not get anything.

Is it possible to somehow do a point mutation on a digested insert? or what other method I can use.

Any help will be greatly appreciated. Thanks in advance


You can build PCR primers with offset cutter sites and PCR around the plasmid. The offset cutter then is used to eliminate the 5' cut site and release a pair of compatible ends which ligate to form the exact sequence you need.