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Takara's Site-Directed Mutagenesis Systems |
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The below-mentioned chard gives you an overview of the different Site-Directed Mutagenesis Systems from Takara. For further information click on the product name.
| Product |
Mutan™-Super Express Km |
LA PCR in vitro Mutagenesis Kit |
Mutan™-Express
Km |
Mutan™-K |
| Order # |
RR022-TK |
RR016-TK |
6090-TK |
6060-TK |
| Principle |
ODA-LA PCR Method |
LA PCR Restriction Enzyme Method |
ODA Method |
Kunkel Method |
| Description |
Takara’s Mutan™-Super Express Km is designed to achieve site-directed mutagenesis in just one day. It is based on Oligonucleotide-directed Dual Amber (ODA) method, and uses LA (Long and Accurate) PCR Amplification |
The LA PCR in vitro Mutagenesis Kit is an improved system to introduce a series of site-directed mutations into long DNA fragments cloned into pUC or pUC-derived vectors containing the pUC multiple cloning site. This system takes advantage of LA (Long and Accurate) PCR technology by including the Takara LA Taq™-enzyme and LA Buffer II. |
Takara’s Mutan™ -Express Km is designed to efficiently introduce site-directed mutations. Using the simplified procedure, a desired mutation can be introduced in
3 days without ssDNA isolation. |
This system is based on the oligonucleotide-directed mutagenesis method developed by Kunkel. The kit allows the introduction of mutations such as base substitutions, deletions or insertions into a DNA fragment cloned into M13 phage or related phagemid vectors. Site-directed mutagenesis is a powerful technique which is often used to study the structure and function of genes and proteins. |
| Efficiency |
> 80% |
> 60% |
70 - 95 % |
60 - 70 % |
| Vectors |
pKF18k-2/19k-2 |
pUC18/19 (118/119) |
pUC18/19 (118/119) |
vectors capable of preparing ssDNA |
| Host strains |
sup° |
any strains |
sup°, mutS |
dut-,ung-,F‘ |
| Preparation of ssDNA |
No preparation |
No preparation |
No preparation |
Should be prepared |
| Time Required |
1 day |
2 - 3 days |
2 - 3 days |
4 - 5 days |
| Notes |
Transformation only using PCR products
(< 2 kb DNA fragments) |
No need for specific host strains |
No need to prepare ssDNA |
Any vectors capable of ssDNA |
| Quantity |
20 reactions |
10 reactions |
20 reactions |
20 reactions |
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Mutan™-Super Express Km
Description
Takara’s Mutan™-Super Express Km site-directed mutagenesis kit is based on ODA method (Oligonucleotide-directed 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.
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Principle
The principle of the Mutan™-Super Express Km is illustrated in Figure 1. Mutan™-Super Express Km utilizes the  pKF18k-2/19k-2 vector, same as Mutan™-Express Km (6090-TK) 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 is 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 site-specific 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.
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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) |
10µl |
| 8. pKF19k-2 DNA (0.5 µg/µl) |
10 µl |
| 9. E. coli MV1184 (10% glycerol solution) |
100µl |
Reagents required but not supplied
- E. coli MV1184 Competent Cells (Order#9055-TK) or E. coli MV1184 Electro Cells (Order#9025-TK)
- Synthetic mutagenic oligonucleotides (phosphorylated at 5′-termini)
- Antibiotics (kanamycin) (Order# 0408-10GAM)
- Medium and plates
Order Information, Shipping & Storage
| Order Information |
| order # |
description |
amount |
| RR022-TK |
Mutan™-Super Express Km for site-directed mutagenesis |
Kit |
| 9055-TK |
E. coli MV1184 Competent Cells |
10 x 100 µl |
| 9025-TK |
E. coli MV1184 Electro Cells |
10 x 50 µl |
| store at -20°C, E. coli MV1184 supplied in the kit must be stored at -80°C after delivery; E. coli MV 1184 at -80°C; shipped on dry ice |
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LA PCR in vitro Mutagenesis Kit
Description
This kit is an improved system to introduce a site-directed mutation into long DNA cloned into pUC vectors (pUC 18/19, 118/119) or pUC derived vectors containing the pUC multiple cloning site. The kit utilizes a researcher-designed primer to introduce the desired mutation in the target sequence, and kit-supplied primers MUT 1 to 6, M13 M4, M13 RV. This combination results in a site-specific mutant which is easily introduced by only preparing one primer, and without the need for repeated bacterial transformations. As based on LA (Long and Accurate) PCR technology using the improved enzyme, TaKaRa LA Taq™, this kit provides the high fidelity, and introduction of mutations into a longer DNA can be achieved.
Principle
A site-directed mutation is introduced through the following protocol (Fig. 2).
1) Insert target DNA into multicloning site of pUC vectors. Choose one of MUT Primers to destroy a  restriction site based on the direction of R1 Primer (primer for introducing a mutation) and the restriction site used for DNA insertion. Perform the first PCR by the combination of R1 Primer and M13 Primer RV (or M13 Primer M4), MUT Primer and M13 Primer M4 (or M13 Primer RV) separately in two tubes.
2) After elimination of excess amount of primers, amplified products are mixed, heat denatured and annealed.
3) Add TaKaRa LA Taq™ to complete heterogeneous double strand.
4) Perform the second PCR by using M13 Primer M4 and M13 Primer RV, which will result in two types of the amplified products (a) and (b).
5) Digest the amplified products with two restriction enzymes, one of which should recognize the site (X) that had been destroyed by MUT Primer and the other should recognize the appropriate site (Y) within the multicloning site.
6) Reclone the digested fragment into the vector digested with the same two restriction enzymes. Only the fragment (a) which contains the mutation introduced by R1 Primer sequence will be recloned.
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Kit components
| 1. MUT 1 Primer [EcoR I, Sac I] (2.5 pmol/µl) |
10 µl |
2. MUT 2 Primer [BamH I, Xba I, Sal I (Acc I, Hinc II)]
(5 pmol/µl) |
10 µl |
| 3. MUT 3 Primer [Sph I, Hind III] (2.5 pmol/µl) |
10 µl |
| 4. MUT 4 Primer [Sph I, Hind III] (2.5 pmol/µl) |
10 µl |
5. MUT 5 Primer [BamH I, Xba I, Sal I (Acc I, Hinc II)]
(5 pmol/µl) |
10 µl |
6. MUT 6 Primer [EcoR I, Sac I] (2.5 pmol/µl)
MUT Primers are designed to anneal within the multiple cloning site of pUC vectors with one base mismatch in the recognition sequence of the restriction enzymes indicated with each primer respectively. |
10 µl |
| 7. M13 Primer M4 (10 pmol/µl) |
15µl |
| 8. M13 Primer RV (10 pmol/µl) |
15µl |
| 9. TaKaRa LA Taq™ (5 units/µl) |
15µl |
| 10. 10 × LA PCR Buffer II (Mg2+ plus)* |
175 µl |
| 11. 10 × Ex Taq Buffer** |
175 µl |
| 12. dNTP Mixture (ea. 2.5 mM) |
400 µl |
| 13. Control Template (1 ng/µl) |
10 µl |
| 14. Control Primer (2.5 pmol/µl) |
10 µl |
| 15. EcoR I (for control experiment; 8-20 units/µl) |
10 µl |
| 16. 10 × EcoR I buffer (for control experiment) |
20 µl |
*for amplification of ≥ 2 kbp DNA fragment
**for amplification of < 2 kbp DNA fragment
Reagents required but not supplied
R1 Primer designed to introduce mutation is not included. User must design and prepare desired R1 Primer.
Order Information, Shipping & Storage
| Order Information |
| order # |
description |
amount |
| RR016-TK |
LA PCR in vitro Mutagenesis Kit for site-directed mutagenesis |
Kit |
| store at -20°C; shipped on dry ice |
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Mutan™-Express Km
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. It is helpful in the study of structure and of function of genes and proteins.
Principle
The principle of the ODA method is shown in Figure 3.
Containing dual
amber mutations on kanamycin-resistant
gene, pKF 18k-2/19k-2 can be propagated only in supE host strains (e.g. JM109).  The target DNA fragment to be mutated is cloned into the multiple cloning site of pKF 18k-2/19k-2. The plasmid DNA is denatured by heat treatment to prepare single-stranded DNA. Oligonucleotides 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 a supE/mutS strain, then DNA starts 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.
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Kit components
Enzyme/Oligo set (Order# 6090-TK) 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 (Order# 6091-TK)
| 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 (tetr)/F' [traD36, proAB+, lacIq, lacZ ΔM15]
** Δ(lac-proAB),ara,rpsL,thi(φ80 lacZ ΔM15), Δ(srl-recA)306:: Tn10 (tetr)/ F'[traD36,proAB+, lacIq, lacZΔM15]
Reagents required but not supplied
- Competent Cells (E. coli BMH71-18 mutS Competent Cells (Order# 9054-TK) and E. coli MV1184 Competent Cells (Order# 9055-TK)) or ElectroCells (E. coli MV1184 Electro Cells(Order# 9025-TK))
- Synthetic mutagenic oligonucleotides (phosphorylated at 5'-termini)
- Antibiotics (kanamycin) (Order# 0408-10GAM)
- Medium and plates
Order Information, Shipping & Storage
| Order Information |
| order # |
description |
amount |
| 6090-TK |
Mutan™-Express Km for site-directed mutagenesis (Enzyme/Oligo set) |
Kit |
| 6091-TK |
Mutan™-Express Km for site-directed mutagenesis (Vector/Host set) |
Kit |
| 9055-TK |
E. coli MV1184 Competent Cells |
10 x 100 µl |
| 9025-TK |
E. coli MV1184 Electro Cells |
10 x 50 µl |
| 9054-TK |
E. coli BMH71-18 mutS Competent Cells |
10 x 100 µl |
| store Enzyme/Oligo set at -20°C, Vector/Host set at -80°C (avoid repeated freeze-thaw cycles); E. coli at -80°C; shipped on dry ice |
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Mutan™-K
Description
This system is based on the oligonucleotide-directed mutagenesis method developed by Kunkel 1, 2). The kit allows the introduction of mutations such as base substitutions, deletions or insertions into a DNA fragment cloned into M13 phage or related phagemid vectors. Site-directed mutagenesis is a powerful technique which is often used to study the structure and function of genes and proteins.
Principle
The principle of the Kunkel mutagenesis method is shown in Figure 4. The target DNA fragment to be mutated is cloned into the multiple cloning site of M13mp18 or M13mp19. Single-stranded phage DNA (ssDNA) is prepared using a bacterial host  strain such as CJ236 for propagation of the M13 phage. E. coli CJ236 is dut– and ung–, and is deficient for the enzymes dUTPase (Dut) and uracil-N glycosylase (Ung). A deficiency of dUTPase leads to the production of elevated levels of dUTP which in turn leads to the misincorporation of deoxyuridine into DNA in place of thymidine. A deficiency of uracil-N glycosylase makes the bacteria unable to remove the uracil residues from the DNA. Thus, M13 phage DNA prepared from phage propagated in CJ236 cells contains deoxyuridine in place of some of the thymidine residues. A synthetic mutagenic oligonucleotide, which is complementary to the target DNA sequence and contains one or more mismatched bases at the site of the desired mutation, is then annealed to the deoxyuridine-containing ss template DNA. The annealed oligo is extended using T4 DNA polymerase to generate a complete complementary strand of DNA, and then the ends are ligated using E. coli DNA ligase. The DNA is used to transform E. coli strain BMH71-18 mutS which is deficient in the ability to repair mismatched DNA and, since it is ung+, it is capable of removing uridines from DNA. The original uridine-containing strand is degraded. The newly synthesized strand which contains the mutated base(s) and does not contain deoxyuridine survives and is replicated to produce intact mutated plasmid. This procedure produces a very low percentage of non-mutated background clones.
This method is applicable to phagemid vectors which can be recovered as single-stranded DNA, such as pUC118 and pUC119, although the mutation frequency may be 5 to 10% higher when an M13 phage vector is used. |
Kit components
Mutan™-K Enzyme Set (Order# 6060-TK) 20 reactions
| 1. Annealing Buffer |
20 µl |
| 2. Extension Buffer |
500 µl |
| 3. E. coli DNA Ligase (60 units/µl) |
20 µl |
| 4. T4 DNA Polymerase (1 unit/µl) |
20 µl |
5. Control ssDNA Solution A (0.2 pmol/µl)
Single-stranded M13mp11 am16 contains |
5 µl |
6. Control ssDNA Solution B (0.2 pmol/µl)
Single-stranded pUC119 am 16 phagemid DNA |
5 µl |
7. Control Oligonucleotide (1 pmol/µl)
5′phosphorylated 16 mer (5′- pGGTTTTCCCAGTCACG-3′) |
5 µl |
Order Information, Shipping & Storage
| Order Information |
| order # |
description |
amount |
| 6060-TK |
Mutan™-K Enzyme Set for site-directed mutagenesis |
Kit |
| store at -20°C; shipped on blue ice |
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Literature
- Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA, 82, 488-492.
- Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Methods in Enzymology, 154, 367-382.
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| ©2007 MoBiTec GmbH • All rights reserved • www.mobitec.com |
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