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Products >  PCR_RT-PCR_Real-Time_qPCR >  Mutagenesis_Reagents >  Transformer_Site-Directed_Mutagenesis_Kit

Site Directed Mutagenesis Kit—Transformer

The Transformer Mutagenesis Kit is a highly efficient system for performing in vitro site-directed mutagenesis (1). Specific mutations—base substitutions, deletions, or insertions—can be introduced into a target gene or region cloned into virtually any double-stranded plasmid with a unique restriction site and a bacterial selection marker (2). This kit can also be used to generate unidirectional nested deletions using an alternative procedure (3).

The Transformer Method

The Transformer Kit uses two oligonucleotide primers that are simultaneously annealed to one strand of a denatured double-stranded template. One primer introduces the desired mutation and the other mutates the unique restriction site in the plasmid, creating a new restriction site or eliminating the site completely. Elongation by T4 DNA polymerase, which lacks strand displacement activity, results in the incorporation of both mutations in the same newly synthesized strand. The DNA is then digested with a restriction enzyme that cuts at the original restriction site. The uncut, mutated DNA will transform E. coli more efficiently than the linear DNA with no mutations.

E. coli BMH 71-18 mutS, which is mismatch repair deficient, is used to propagate the mutated plasmid. Two rounds of DNA digestion and transformation ensure that a very high frequency of transformants carry the mutated plasmid, which nearly always contains both mutations—the desired mutation and the selection mutation (1, 4).

The Transformer Site-Directed Mutagenesis Kit contains enough reagents for 30 mutagenesis reactions, including 10 control reactions. You must supply the selection and mutagenic primers and the vector. The selection primer must contain a mutation that will eliminate a unique restriction site located on the plasmid or will convert the unique restriction site into another unique site. Multiple rounds of mutagenesis may then be performed on the gene of interest without recloning.

Chemically competent BMH 78-18 mutS E. coli cells are available separately for use with the Transformer Mutagenesis Kit. These cells are mismatch-repair deficient.

At-A-Glance Documents Images & Data

Features

  • Obtain high mutagenesis efficiencies
  • Use any double-stranded plasmid
  • No subcloning required

Applications

  • Site-directed mutagenesis

References

  1. Deng, W. P. & Nickoloff, J. A. (1992) Anal. Biochem. 200(1):81–88.
  2. Haught, C. et al. (1994) BioTechniques 16(1):47–48.
  3. Zhu, L. & Holtz, A. (1996) Methods Mol. Biol. 57:119–137.
  4. Zhu, L. (1995) Methods Mol. Biol. 57:13–29.

Additional Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


 
 
Products
Cat. # Product Package Size Price # of Units Select
630701 BMH 71-18 mutS Competent Cells 5 x 0.2 mL $230.00
630702 Transformer™ Site-Directed Mutagenesis Kit Each $445.00
 

 

 

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