Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%. In addition, up to 6 different mutations can be introduced in one reaction without causing any fortuitous change in the vector backbone. Using this strategy, we have introduced 32 S/T➔A substitutions in the N-terminus head and 13 changes in the C-terminus tail domain of vimentin.
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