The Site-Specific Mutagenesis Kit is used to introduce point mutations, and deletion or insertion into constructed genes. The site-specific mutagenesis is performed using Pfu Plus DNA polymerase by amplifying whole plasmid DNA with mutant complementary primer pair. Pfu Plus DNA polymerase replicates both strands of double-stranded DNA without displacing the mutant primers.The basic procedure utilizes a circular double-stranded plasmid DNA (dsDNA) with an insert of interest, and two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by DNA polymerase. Incorporation of the oligonucleotide primers generates a mutated plasmid containing taggered nicks. Following temperature cycling, the product is digested with Dpn I restriction endonuclease (restriction sequence: 5'-GmATC-3') which is specific for methylated and hemimethylated parental dsDNA. The plasmid DNA isolated from dam+ E.coli strain is susceptible to Dpn I digestion. The Dpn I digested PCR product containing desired mutations is then transformed into E.coli competent cells.Highly accurate Pfu Plus DNA polymerase and the low number of thermal cycles all contribute to the high efficiency and the decreased potential for generating unwanted mutations during the reaction.
Pfu Plus Polymerase is effective in the amplification of smaller than 12 kbp PCR products. All the components, except competent cells, are included in the kit including Pfu Plus Polymerase, Dpn I restriction enzyme, buffers, dNTP mix, control vector, and control primers. The reagents are sufficient for 50 separate reactions.
- Pfu Plus DNA polymerase : 250 unit (5 unit/㎕)
- 10x reaction buffer : 1 ml
- Dpn I restriction enzyme : 500 unit (10 unit/㎕)
- Control primer :
- Sense primer #1 10 μl (10 pmol/㎕)
- Antisense primer #2 10 μl (10 pmol/㎕)
- pBS control plasmid : 50 ng (5 ng/㎕)
- dNTP mix : 0.5 ml (2.5 mM each)
- Store at -20℃.
- The performance of Site-Specific Mutagenesis Kit is monitored routinely on a lot number. The pBS control plasmid containing β-galactosidase gene under the control of lac promoter is used to test the efficiency of mutant generation.
Step 1) PCR with control primer pair (#1,2) using Pfu Plus DNA Polymerase,
Step 2) Dpn I digestion of PCR products to remove template DNA,
Step 3) Transformation into host cells, and screen mutation rate by blue/white assay.
The mutated pBS plasmid contains a stop codon (TAA) at the position where a glutamine codon (CAA) would normally appear in the β-galactosidase. The white phenotype on IPTG/X-gal plate indicates that mutation is normally introduced into control β-galactosidase gene. The overall efficiency of mutagenesis is approximately 98%.
- Point mutagenesis
- Introduction of base insertion and deletion