DpnI
Detailní popis
Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.
Highlights
- Superior quality – stringent quality control and industry leading manufacturing process
- Convenient color-coded Five Buffer System
- Includes universal Tango buffer for double-digestions
- BSA premixed in reaction buffers
- Wide selection of restriction endonuclease specificities
Applications
- Molecular cloning
- Restriction site mapping
- Genotyping
- Southern Blot
- Restriction fragment length polymorphism (RFLP)
- SNP
Note
DpnI requires the presence of N6-methyladenine within the recognition sequence to cleave DNA. DNA purified from a dam+ strain will be a substrate for DpnI. DpnI will only cleave fully-adenomethylated dam sites. Hemi-adenomethylated dam sites DpnI cleaves 60X more slowly. DpnI, Bsp143I, and MboI all recognize the same sequence, but have different methylation sensitivities and cleavage sites. Assayed using pBR322 DNA.
For methylation sensitivity refer to product specifications.
| Conditions for 100% Activity |
|
| Double Digestion | Perform double digestion using DoubleDigest. |
| Hazardous | No |
| Isoschizomers | Search for commercial isoschizomers using REsearch. |
| Storage Buffer | DpnI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/mL BSA, and 50% (v/v) glycerol. |
| Storage Condition | -20 C |
Reaction conditions
| Recommended buffer for 100% activity | Optimal temperature | Enzyme activity in Thermo Scientific buffers, % | Tango buffer for double digestion | |||||
|---|---|---|---|---|---|---|---|---|
| B (blue) 1X |
G (green) 1X |
O (orange) 1X |
R (red) 1X |
Tango (yellow) 1X / 2X |
||||
| Tango | 37°C | 100 | 100 | 50-100 | 50-100 | 100 | 50-100 | 1X or 2X |
Methylation Effects
- Dam: does not cut dam - DNA.
- Dcm: never overlaps - no effect.
- CpG: may overlap - no effect.
- EcoKI: never overlaps - no effect.
- EcoBI: may overlap - effect not determined.
| Methylation type | Sequence | Cleavage effect |
|---|---|---|
| CpG | 5'...Gm6A Tm5C G...3'3'... C Tm6A Gm5C...5' |
Cleaves only Dam methylated DNA |
Dam (GATC) |
Gm6ATC |
Cleaves only Dam methylated DNA |
Cleavage efficiency close to the termini of PCR fragments
| bp from the recognition site to fragment end | ||||
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 |
| 50-100 | ||||
Number of recognition sites in DNA molecules
| Lambda | ΦX174 | M13mp18/19 | pBR322 | puc18/19 | pUC57 |
|---|---|---|---|---|---|
| 116 | 0 | 7 | 22 | 15 | 15 |
| pTZ19R/U | pTZ57R | pBluescriptIIKS(±) | pBluescriptIISK(±) | pACYC177 | pACYC184 |
| 15 | 15 | 15 | 15 | 22 | 15 |
New sites generated by ligation
Newly generated recognition sites resulting from ligation of blunt DNA ends
| Recognition sequence | Second restriction enzyme | Restriction enzymes cleaving the newly generated recognition sequence |
|---|---|---|
GA^TC |
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