Description
DpnI restriction enzyme for mutagenesis
DpnI is a restriction endonuclease that cleaves only methylated and hemimethylated DNA (recognition site Gm6ATC). It is used to digest the parental plasmid template after site-directed mutagenesis or amplification, efficiently removing the original DNA while leaving the newly synthesised, unmethylated product intact.
This recombinant fast-digest enzyme cuts DNA accurately within 5–15 min and suits plasmid DNA, PCR products and genomic DNA. It has 100% activity in the supplied universal buffer, so multiple fast-digest restriction enzymes can be used together in one reaction. The supplied 10× Color Buffer contains red and yellow tracking dyes for direct gel loading: in a 1% agarose gel the red dye runs with ~2,500 bp and the yellow dye with ~10 bp double-stranded DNA. The enzyme is heat-inactivated at 80°C for 20 min. For research use only.
Key Features
| Fast | Digestion is complete in as little as 5–15 min. | |
| Methylation-specific | Cleaves only methylated and hemimethylated GATC sites, leaving unmethylated PCR product intact. | |
| Universal buffer | Full activity in the universal buffer simplifies multi-enzyme digestion. | |
| Color buffer included | Built-in tracking dyes allow digests to be loaded directly onto gels. |
Specifications
| Enzyme type | Recombinant fast-digest restriction endonuclease |
| Recognition site | Gm6ATC (methylated or hemimethylated only) |
| Digestion time | 15 min (plasmid DNA); 30–60 min (genomic DNA) at 37°C |
| Applications | Digestion of genomic and plasmid DNA; removal of plasmid template after amplification |
| Heat inactivation | 80°C for 20 min |
| Shelf life | 2 years |
Components
| Components | 50 rxns |
| DpnI | 50 μl |
| 10× Universal Buffer | 1 ml |
| 10× Universal Color Buffer | 1 ml |
Storage
Store at -30 ~ -15°C.
- Set up the digestion
- Prepare the reaction as follows. Scale the volume up or down proportionally as required.
| Components | Plasmid DNA | Genomic DNA |
| 10× Universal Buffer or 10× Universal Color Buffer | 2 μl | 5 μl |
| DpnI | 1 μl | 5 μl |
| DNA | ≤1 μg | ≤5 μg |
| ddH2O | Up to 20 μl | Up to 50 μl |
- Mix
- Mix by flicking the tube, then centrifuge briefly to collect the liquid at the bottom.
- Incubate
- Incubate at 37°C for 15 min (plasmid DNA) or 30–60 min (genomic DNA).
- Inactivate
- Heat-inactivate DpnI at 80°C for 20 min.
Double or multiple digestions
- Use the enzyme amounts in the table above for each enzyme and scale the volume as needed. Adjust the buffer volume so the final concentration is 1×.
- Keep the total enzyme volume ≤1/10 of the reaction volume.
Methylation sensitivity
| Dam | Dcm | CpG | EcoKI | EcoBI |
| Not sensitive | Not sensitive | Impaired | Not sensitive | Impaired |
Q1: Which DNA does DpnI cut?
- DpnI cleaves only methylated and hemimethylated GATC sites. Plasmid DNA purified from a dam+ strain is a substrate, while newly synthesised, unmethylated PCR product is left intact.
Q2: How long should I digest?
- Incubate at 37°C for 15 min for plasmid DNA or 30–60 min for genomic DNA. Avoid digesting for more than 3 h, which may cause star activity.
Q3: Can I combine DpnI with other restriction enzymes?
- Yes. Use the recommended amount of each enzyme, adjust the buffer to a final 1× concentration and keep the total enzyme volume ≤1/10 of the reaction volume.
Q4: How do I inactivate DpnI?
- Incubate at 80°C for 20 min.
