A) Prepare template DNA and primers
Subclone the DNA to be mutagenized into vector
Prepare template DNA by miniprep
Oligo primers is synthesized by GIBCO

(B) First PCR amplification
Amplify the template DNA and generate blunt-end fragment
Purify the fragments by agarose gel electrophoresis

(C) Second PCR amplification
Combine each amplified fragment with each flanking primer
PCR and analyze the reaction

(D) Subclone fragments and check the mutation
Digest the DNA fragment with restriction endonuclease
Subclone into vector
Confirm mutation by sequencing