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Transformer™ Site-Directed Mutagenesis Kit

 
 
 
 

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If you do not see the Certificate of Analysis for your specific lot, please follow this link.
 
 
 
 

Citations

 
Product Name Catalog Number Authors Title Year Journal
Transformer Site-Directed Mutagenesis Kit 630702 Reiko Murai-Takebe, Tetsuya Noguchi, Takeshi Ogura, Toshiyuki Mikami, Kazunori Yanagi, Kenjiro Inagaki, Hiroshi Ohnishi, Takashi Matozaki, and Masato Kasuga Ubiquitination-mediated Regulation of Biosynthesis of the Adhesion Receptor SHPS-1 in Response to Endoplasmic Reticulum Stress March 2004 J. Biol. Chem., Mar 2004; 279: 11616 - 11625
Transformer Site-Directed Mutagenesis Kit 630702 Haiqiang Mai, W. Stratford May, Fengqin Gao, Zhaohui Jin, and Xingming Deng A Functional Role for Nicotine in Bcl2 Phosphorylation andSuppression of Apoptosis January 2003 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 3, Issue of January 17, pp. 1886-1891, 2003
Transformer Site-Directed Mutagenesis Kit 630702 Verkhusha, V. V., Otsuna, H., Awasaki, T., Oda, H., Tsukita, S. and Ito, K. An Enhanced Mutant of Red Flourescent Protein DsRed for Double Labeling and Developmental Timer of Neural Fiber Bundle Formation June 2001 J. Biol. Chem. 276(32):29621-29624
Transformer Site-Directed Mutagenesis Kit 630702 Kaetzel, M. A., Mo, Y. D., Mealy, T. R., Campos, B., Bergsma-Schutter, W., Brisson, A., Dedman, J. R. & Seaton, B. A. Phosphorylation Mutants Elucidate the Mechanism of Annexin IV-Mediated Membrane Aggregation March 2001 Biochemistry 2001(40):4192-4199
Transformer Site-Directed Mutagenesis Kit 630702 Armand, S., Andrews, S. R., Charnock, S. J. & Gilbert, H. J. Influence of the Aglycone Region of the Substrate Binding Cleft of Pseudomonas Xylanase 10A on Catalysis May 2001 Biochemistry 2001(40):7404-7409
Transformer Site-Directed Mutagenesis Kit 630702 Yakovlev, S., LItvinovich, S., Loukinov, D. & Medved, L. Role of the b-strand Insert in the Central Domain of the Fibrinogen g-Module November 2000 Biochemistry 39(51):15721-15729
Transformer Site-Directed Mutagenesis Kit 630702 Vince, J. W., Carlsson, U. & Reithmeier, R. A. F. Localization of the Cl-/HCCO3-Anion Exchanger Binding Site to the Amino-Terminal Region of Carbonic Anhydrase II October 2000 Biochemistry 39(44):13344-13349
Transformer Site-Directed Mutagenesis Kit 630702 Ling, S., Woronuk, G., Sy, L., Lev, S. & Braun, A. P. Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase September 2000 J. Biol. Chem. 275(39):30683-30689
Transformer Site-Directed Mutagenesis Kit 630702 Relaix, F., Wei, X., Li, W., Pan, J., Lin, Y., Bowtell, D. D., Sassoon, D. A. & Wu, X. Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis February 2000 Cell Biology 97(5):2105-2110
Transformer Site-Directed Mutagenesis Kit 630702 Drosopoulos, J. H. F., Broekman, M. J., Islam, N., Maliszewski, C. R., Gayle III, R. B. & Marcus, A. J. Site-Directed Mutagenesis of Human Endothelial Cell Ecto-ADPase/Soluble CD39: Requirement of Glutamate 174 and Serine 218 for Enzyme Activity and Inhibition of Platelet Recruitment May 2000 Biochemistry 39(23):6936-6943
Transformer Site-Directed Mutagenesis Kit 630702 Hruz, P. W. & Mueckler, M. M. Cysteine-Scanning Mutagenesis of Transmembrane Segment 11 of the GLUT1 Facilitative Glucose Transporter July 2000 Biochemistry 39(31):9367-9372
 

The Transformer Mutagenesis Kit is a high efficiency system for performing in vitro site-directed mutagenesis (1).

Specific mutations—base changes, 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 which 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 reagents sufficient for a total of 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 enzyme 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 E. coli BMH 78-18 mutS cells are available separately for use with the Transformer Mutagenesis Kit. These cells are mismatch-repair deficient.

 
 
 
 

Figure 1. The Transformer Site-Directed Mutagenesis Kit method.

 
 
 
 

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.

    Components

    E. coli BMH 71-18 mutS Strain
    Annealing Buffer
    Synthesis Buffer
    T4 DNA Polymerase
    T4 DNA Ligase
    Control Template pUC19M
    Two Control Primers (mutagenic & selection)
    Control Restriction Enzyme

    Storage Conditions

    Store E. coli BMH 71-18 mutS at -70C
    Store all other components at -20C