Description

Takara's Mutan™-Super Express Km site directed mutagenesis kit is based on ODA method (Oligonucleotidedirected Dual Amber method) utilizing the advantage of LA (Long and Accurate) PCR technology and is designed to achieve site-directed mutagenesis just in one day.

Storage

E. coli MV1184: –80°C
   E. coli MV1184 supplied in the kit must be stored at –80°C after delivery.
All other components: –20°C

Kit components

1. TaKaRa LA Taq™ (5 units/µl)	10 µl
2. 10 × LA PCR Buffer II (Mg2+ plus)	100 µl
3. dNTP mixture (ea. 2.5 mM)	160 µl
4. Selection Primer (5 pmol/µl)	20 µl
5. Control Synthetic Oligonucleotide (5 pmol/µl)	5 µl
6. Control dsDNA Solution (pKF19kM) (10 ng/µl)	5 µl
7. pKF18k-2 DNA (0.5 µg/µl)	10 µl
8. pKF19k-2 DNA (0.5 µg/µl)	10 µl
9. E. coli MV1184 (10% glycerol solution)	100 µl

Reagents required but not supplied
• E.coli MV1184 Competent Cells (Cat.#9055) or E.coli MV1184 Electro Cells (Cat.#9025)
• Synthetic mutagenic oligonucleotides (phosphorylated at 5′-termini)
• Antibiotics (kanamycin)
• Medium and plates

Principle

The principle of the Mutan™-Super Express Km is illustrated in Figure 1. Mutan™-Super Express Km utilizes the pKF18k-2/19k-2, same as Mutan™-Express Km (Cat.# 6090) does. Since the vector contains dual amber mutations on the kanamycin-resistant gene, obtained transformants do not show kanamycin resistance when introduced in the sup⁰ host strain, such as MV1184. After the target for mutagenesis is produced by cloning into the pKF18k-2/19k-2 vectors, PCR is performed using the oligonucleotide containing the desired mutation and a selection primer to revert the amber mutations on the kanamycin-resistant gene as PCR primers. With this PCR, the sequence between the two primers are amplified, generating Mutagenic-Selection DNA. As this amplified DNA would be also used as a PCR primer simultaneously, the polymerase reaction further progresses and nicked double-stranded plasmid can be yielded. Utilizing the advantage of LA PCR technology, Mutan™-Super Express Km performs higher fidelity and longer PCR in extension process of plasmid strands. When this nicked DNA is transformed into E. coli MV1184 (sup⁰ strain), the nick is repaired and then transformants containing a desired sitespecific mutation can be grown on the medium containing kanamycin. Thus Mutan™-Super Express Km achieves the introduction of mutation at > 80% efficiency just in one day by following a simple procedure.



Figure 1 Principle of ODA-LA PCR method