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EZchange™ Multi Site-directed Mutagenesis core Kit

No Product Name Cat.# Conc Size Price(₩) Add
1 EZchange™ Multi Site-directed Mutagenesis core Kit EZ024S - 10 rxns ₩ 250,000 추가
2 EZchange™ Multi Site-directed Mutagenesis core Kit EZ024M - 20 rxns ₩ 450,000 추가
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Product description

EZchange™ Multi site-directed mutagenesis Kit is designed to create multiple site-directed mutations in plasmid DNA. The product consists of three processes: amplification, DpnI digestion, and transformation. Step 1, Mutagenic primers are annealed to denatured template plasmid. Each primer has the desired sequence changes. DNA polymerase extends the oligonucleotides and ligase seals the DNA strands creating a replicated plasmid possessing the desired mutation. This process is repeated in PCR amplification for 30 cycles. In step 2, PCR amplification reaction is treated Dpn I endonuclease, it cleaves methylated and hemi-methylated DNA. In step3, Dpn I reaction mixture is transformed into competent cells, where ssDNA is converted into dsDNA. This simple and quick procedure provides high mutagenesis efficiency greater than 50%, even with multiple mutations.

 

Kits contents

- EZchange™ Multi Enzyme mixture

- EZchange™ Multi 10X Reaction buffer

- dNTP mixture (2 mM each)

- EZchange™ Multi Control plasmid (50 ng/μl)

- EZchange™ Multi Control primer mixture (10 pmol/μl each of three primers)

- Dpn I restriction endonuclease (20 units/μl)

- Sterile water

 

Reagents not provided in the Kit

- DH5α Chemically Competent E. coli

- X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)

- IPTG (Isopropyl β-D-1-thiogalactopyranoside)

 

Storage conditions

- 10X reaction buffer, DH5α competent cell: -80 

- All other component: -20

 

Standard reaction conditions

1. Combine mutagenesis reaction component as described below then mix gently.

Reaction component

Final conc.

Sample

Control

10X reaction buffer

1X

2.5 μl

2.5 μl

Template plasmid

50 ng

x μl

1 μl

Mutagenic primers

Each 10 pmol

x μl

1 μl

dNTP mixture

0.2 mM

2.5 μl

2.5 μl

Enzyme mixture

-

1 μl

1 μl

Sterile water

-

Up to 25 μl

17 μl

 

2. Run thermal cycling reaction as indicated below.

  * For the control reaction, use an 8 minute extension time.

Temperature

Time

Cycles

95

1 minute

1 cycle

95

1 minute

30 cycles

55

1 minute

65

2 minute /kb of plasmid length

8 minute for the control reaction

 

3. Add 1 μl DpnI restriction enzyme to the reaction mixture directly and incubate 30 min at 37 

 

4. Transfer 2 μl of DpnI treated reaction mixture into DH5α competent cells for transformation.

 

Note

- Avoid defrost cycle of reaction buffer and dNTP mixture. We recommend make small aliquots, each for 

  a single use.

- Template plasmids should be purified from dam+ E. coli strains. Plasmid DNA isolated from the exceptional   dam- E.coli strains, including JM110 and SCS110 is not suitable, since they are not digested by Dpn I. 

- This kit is not recommended for use with template plasmid greater than 5 kb. 

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