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.
- 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)
- DH5α Chemically Competent E. coli
- Sterile water
Reagents not provided in the Kit
- X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
- IPTG (Isopropyl β-D-1-thiogalactopyranoside)
- 10X reaction buffer, DH5α competent cell: -80℃
- All other component: -20℃
- 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.