Takara
PCR How to Select the Best PCR Enzyme Guide to Takara PCR Polymerases High Fidelity PCR High Speed PCR High Performance PCR DNA Bisulphite Modification Real Time (qPCR) Kits General PCR Products RT-PCR and RACE Food Pathogen Detection & Screening Kits PCR Probes & Primer Sets Molecular Biology Cloning, Mapping & cDNA Synthesis Gene Transfer & Expression RNA/Protein/DNA Extraction Nucleic Acids Electrophoresis Instrumentation DNA Labeling & Detection Kits Sequencing, Mutagenesis & Mutation Analysis Molecular Biology Reagents Restriction Enzymes Modifying Enzymes: Kinases & RT Modifying Enzymes: Phosphatases Modifying Enzymes: Ligases Modifying Enzymes: DNA Polymerase Modifying Enzymes: RNA Polymerase Modifying Enzymes: Nucleases Modifying Enzymes: Rnase Inhibitor DNA Microarray Accessories Cell Biology Apoptosis Antibodies Bone Research Products Cell Adhesion & Extracellular Matrix Cell Differentiation Kit Cell Proliferation & Viability Signal Transduction Viral & Microbial Detection Protein Research Recombinant Protein Folding & Expression C-Terminal Isolation & Analysis N-Terminal Deblocking & Analysis Protein Fragmentation Protease Inhibition Glycobiology Glycobiology Kits Related Enzymes Order Products
About Takara Bio USA Clontech-Takara Bio USA Merger Contact Us Takara Subsidiares and Distributors How to Order Career Opportunities
Press Releases Trade Shows Company News

Mutan™-Express Km

Applications:

  • Site-Directed Mutagenesis Based on the Oligonucleotide-Directed Dual Amber (ODA) Method

Description:

Takara's Mutan™-Express Km kit is designed to efficiently introduce site-directed mutations. Using the simplified procedure, a desired mutation can be introduced in 3 days without ssDNA isolation.

Principle

The oligonucleotide-directed dual amber (ODA) method utilizes the pKF 18k/19k vector containing dual amber mutations on a kanamycin-resistant gene that can only be propagated in a supE host strain. The target DNA to be mutated is cloned into the multiple cloning site of pKF 18k/19k and ssDNA is prepared by heat denaturation. An oligonucleotide containing the desired mutation, along with the selection primer for revision of amber on the kanamycin gene are simultaneously hybridized to the ssDNA. A complementary strand is synthesized by polymerase action. This newly synthesized strand is introduced into the supE/mutS strain and DNA replication proceeds. By selecting DNA which is propagated only in the sup0 strain, the target DNA with the desired mutation is obtained.

Kit Components:

TAK 6090
Annealing Buffer 40 μL
Extension Buffer 60 μL
T4 DNA Ligase (60 U/μL) 20 μL
T4 DNA Polymerase (1 U/μL) 20 μL
Selection Primer (5 pmol/μL) 20 μL
Control dsDNA Solution (50 fmol/μL) 5 μL
Control Synthetic Oligonucleotide Solution (50 pmol/μL) 5 μL
 
Vector Set (Optional) TAK 6091
pKF 18 k-2 DNA(10 OD/mL) 10 μL
pKF 19 k-2 DNA (10 OD/mL) 10 μL
E. coli BMH71-18 mutS* (10% glycerol solution) 100 μL
E. coli MV11 84** (10% glycerol solution) 100 μL
*Δ(lac-proAB), supE, thi-1, mutS 215:: Tn10 (tetr)/F1 [traD36, proAB+, lacIq, lacZ ΔM15]
**Δ(lac-proAB), ara, rpsL, thi (φ 80 lacZ ΔM15), Δ (srl-recA) 306:: Tn10 (tetr)/F1 [traD36, proAB+, lacIq, lacZ ΔM15]

References:

  1. Hashimoto-Gotoh, T. et al. (1995) Gene 152:271-5.
  2. Sambrook, J. et al. (1989) Molecular Cloning (2nd Ed.) 1:74-84.
  3. Sambrook, J. et al. (1989) Molecular Cloning (2nd Ed.) 1:25-31.
  4. Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biology 1:8.1-8.8.
  5. Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biology 1:6.1-6.10.
  6. Dower, W.J. et al. (1988) Nucleic Acids Res. 16:6127.
  7. Zoller, M.J. and Smith, M. (1983) Meth. Enzymol. 100:468.
  8. Hanahan, D. (1983) J. Mol. Biol.166:557.

 

Limited Use Label License: [M22]

Storage:

–20°C

Mutan™-Express Km TAK 6090 20 reactions 276.00
Mutan™-Express Km (Vector/Host Set) TAK 6091 20 reactions 110.00
Product Name Product Number Size Price Qty Add

*All prices in USD