PCRMUT1/27/93

SITE-DIRECTED MUTAGENESIS BY PCR

SUMMARY:

PCR-based, site-directed mutagenesis by PCR was carried out basically as described (PCR Protocols). Two complementary oligonucleotide primers 21 nucleotides in length were created, each containing a point mutation at position 11 (inside primers). These were used seperately as primers for PCR from the wild type cDNA template, along with primers exactly complementary to sequences encompassing convenient restriction sites in the wild type cDNA (outside primers). PCR reactions were denatured for 1 minute at 94^oC, annealed for 1 minute at 37^oC, and extended for 1 minute at 72^oC for 20 cycles. Reaction products were then seperated by agarose gel electrophoreseis and the desired products were purified by Geneclean or Mermaid kits (Bio 101). Products from the two initial reactions were annealed and the full length mutant segment was amplified by PCR using the outside primers. The mutant product was isolated, digested with the appropriate restriction enzymes, and inserted in place of the corresponding wild-type segment in the cDNA. Mutations were comfirmed by sequence.

REACTION 1:

(use 0.5 ml eppendorf tubes, labeled on the TOP)

Template DNA (plasmid with wild-type gene) (20 ng/ul) 5 ul

Mutant (inside) primer 1 (20 pmol/ul) 5 ul

Outside primer 1 (20 pmol/ul) 5 ul

10X Taq polymerase buffer 10 ul

dNTP mix (2mM ea dNTP, contains all dNTP's) 10

Taq polymerase (Cetus) 0.5 ul

sterile dH2O vol to 100 ul

(Run two reactions, one with primer set one, one with primer set two)

Overlay with about 100 ul of paraffin oil.

Run reaction: 3 minutes 94oC, 3 min 37^oC, 3 min 72^oC (1 cycle)

1 min 94, 1 min 37, 1 min 72 (20 cycles)

extension 72 C for 5 minutes

soak 15 C indefinitely

CLEAN UP PRIMARY REACTION PRODUCTS:

Phenol/Chloroform extract reaction products, and ethanol precipitate.

Run on appropriate percentage agarose gel (usually 1-2.5% low melt or mermaid agarose, depending on fragment size).

Excise appropriate bands from gel and purify by geneclean or mermaid.

Elute DNA into 100 ul of water.

REACTION 2:

Product 1 (from REACTION 1)* 10 ul

Product 2 (from REACTION 1)* 10 ul

Outside primer 1 (20 pmol/ul) 5 ul

Outside primer 2 (20 pmol/ul) 5 ul

10X Taq polymerase buffer 10 ul

10X dNTP mix 10 ul

Taq polymerase 0.5 ul

sterile dH2O to 100 ul

Overlay reaction with about 100 ul of paraffin oil.

Run reaction as above.

*If your first reaction worked robustly, use only 1/10 of DNA per 2^o reaction.

CLEAN/DIGEST DNA:

Remove reaction from tube, phenol/chloroform extract, ethanol precipitate.

Digest wild type plasmid and mutation-containing fragment with appropriate enzyme(s).

Run on gel and purify band by geneclean or mermaid, as above.

Run on gel, geneclean/mermaid.

Ligate new insert into rest of wild-type cDNA.