Site-directed Mutagenesis by PCR (CAT#: STEM-MB-0231-WXH)

In brief, point-mutations can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification and the absense of additional modifications.

• Conventional cloning (to introduce or remove restriction sites).
• Mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs).
• Functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context).
• To study changes in protein activity that occur as a result of the DNA manipulation.
• To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property.
• To introduce or remove restriction endonuclease sites or tags.

1. Isolate methylated plasmid with cDNA of Gene X.
2. PCR: Denature plasmid and anneal primers. Primer sequence differs from cDNA by 2 bp where mutation is introduced.
3. Primer extension creates two fragments with a pair mutation. Anneal and extend for several cycles.
4. Digest template
5. Transform product into cells. Screen for positive colonies by digestion with restriction enzyme or screen by DNA sequencing.