Description

Mutan™-Express Km is a site-directed mutagenesis system based on the ODA method (Oligonucleotide-directed Dual Amber method). This kit allows highly efficient site-directed mutation with a simple procedure. This kit is helpful in the study of the structure and of function of genes and proteins.

Storage

Enzyme/Oligo set: -20°C
Vector/Host set: -80°C(Avoid repeated freeze-thaw cycles.)

Kit components

Enzyme/Oligo set (Cat.# 6090) 20 reactions

1. Annealing Buffer	40 µl
2. Extension Buffer	60 µl
3. T4 DNA ligase (60 units/µl)	20 µl
4. T4 DNA polymerase (1 unit/µl)	20 µl
5. Selection Primer (5 pmol/µl)	20 µl
6. Control dsDNA Solution (pKF 19kM dsDNA 50 fmol/µl)	5 µl
7. Control Synthetic Oligonucleotide Solution (50 pmol/µl)	5 µl

Vector/Host set (Cat.# 6091)

1. pKF 18k-2 DNA (10 OD/ml)	10 µl
2. pKF 19k-2 DNA (10 OD/ml)	10 µl
3. E. coli BMH71-18 mutS* (10% glycerol solution)	100 µl
4. E. coli MV1184** (10% glycerol solution)	100 µl

* Δ(lac-proAB), supE, thi-1, mutS 215:: Tn10 (tet^r)/F' [traD36, proAB⁺, lacI^q, lacZ ΔM15]
** Δ(lac-proAB),ara,rpsL,thi(φ80 lacZ ΔM15), Δ(srl-recA)306:: Tn10 (tetr)/ F'[traD36,proAB⁺, lacI^q, lacZΔM15]

Reagents required but not supplied
• Competent Cells (E. coli BMH71-18 mutS Competent Cells and E. coli MV1184 Competent Cells) or ElectroCells (E. coli BMH71-18 mutS Electro Cells and E. coli MV1184 Electro Cells)
• Synthetic mutagenic oligonucleotides (phosphorylated at 5′-termini)
• Antibiotics (kanamycin)
• Medium and plates

Principle

The principle of the ODA method is shown in Figure 1. Containing dual amber mutations on kanamycin-resistant gene, pKF 18k-2/19k-2 can be propagated only in supE host strain (e.g. JM109). The target DNA fragment to be mutated is cloned into the multiple clonig site of pKF 18k-2/19k-2. The plasmid DNA is denatured by heat treatment to prepare the single-strand DNA. Oligonucleotide for the desired mutation and selection primer for reversion of amber on kanamycin gene are simultaneously hybridized to the obtained single-stranded DNA. The complimentary strand is synthesized by polymerase reaction. This newly synthesized strand is introduced into supE/mutS strain, then DNA strarts to replicate without repairing misincorporation. By selecting DNA which is capable of propagation only in sup⁰ strain, the target DNA with a desired mutation is efficiently obtained.



Figure 1 Principle of ODA mutagenesis method