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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

-Gustavo-

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.

-phage434-