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Products >  Molecular_Biology >  Sequencing_Mutagenesis_and_Mutation >  Mutan-Super_Express_Km

Mutan-Super Express Km

Takara's Mutan-Super Express Km Kit is intended for successful site-directed mutagenesis in just one day. This site-directed mutagenesis kit is based on the Oligonucleotide-directed Dual Amber or ODA method and takes advantage of LA (Long and Accurate) PCR.

Principle

The Mutan-Super Express Km utilizes the pKF18k/19k vector system; these vectors contain dual amber stop mutations at their kanamycin-resistance gene. After a target sequence (< 2 kb) is inserted into either of these vectors, PCR is performed using an oligonucleotide containing the desired mutation and a selection primer that reverts the amber mutations on the kanamycin-resistance genes. The sequence between the two primers is then amplified to generate "Mutagenic-Selection DNA." Both strands of this amplified DNA fragment also serve as PCR primers, generating nicked double-stranded plasmid. When this nicked DNA is transformed into E. coli MV1184 (sup0 strain), the nicks are repaired. Transformants containing the desired site-specific mutation can be obtained after kanamycin drug selection. This simple site-directed mutagenesis procedure takes just one day and achieves mutation frequencies greater than 80%.

  At-A-Glance   Documents   Images & Data

Applications

  • Site-directed mutagenesis in one day

Components

LA Taq DNA Polymerase (5 U/µL)10 µL
10X LA PCR Buffer II (Mg2+ plus) 100 µL
dNTP mixture (ea. 2.5 mM) 160 µL
Selection primer (5 pmol/µL)20 µL
Control Synthetic Oligonucleotide (5 pmol/µL)5 µL
Control dsDNA solution (pKF19kM) (10 ng/µL)5 µL
pKF18k DNA (0.5 µg/µL) 10 µL
pKF19k DNA (0.5 µg/µL)10 µL
E. coli MV1184 (10% glycerol solution)*100 µL

*Contains cell stock. Competent cells must be prepared using cultures derived from this stock.

Storage

E. coli MV1184: –80°C 

All other components: –20°C

References

  1. Hashimoto-Gotoh, T. et al. (1995) Gene 152:271-275.
  2. Dower, W.J. et al. (1988) Nucleic Acids Res. 16:6127-6145.
  3. Hanahan, D. (1983) J. Mol. Biol. 166:557-580.



 
 
Products
Cat. # Product Contents Size Price License Units Select
RR022 Mutan™ Super Express Km 20 Rxns $394.00 License Statements
 

 

 
 

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