PagI (BspHI)
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
Low salt, high glycerol (> 5%) concentrations, pH > 8.0 or a large excess of enzyme may result in star activity. PagI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam-, dcm- strain such as GM2163.
For methylation sensitivity refer to product specifications.
| Compatible Ends | AflIII, PscI, DsaI, Eco130I, NcoI. |
| Conditions for 100% Activity |
|
| Digestion of Agarose-embedded DNA | Minimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours. |
| Double Digestion | Perform double digestion using DoubleDigest. |
| Hazardous | No |
| Isoschizomers | Search for commercial isoschizomers using REsearch. |
| Storage Buffer | PagI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 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 |
||||
| O (Orange) | 37°C | 0-20 | 50-100 | 100 | NR | NR | NR | NR |
Methylation Effects
- Dam: may overlap - cleavage impaired.
- Dcm: never overlaps - no effect.
- CpG: never overlaps - no effect.
- EcoKI: never overlaps - no effect.
- EcoBI: may overlap - cleavage impaired.
| Methylation type | Sequence | Cleavage effect |
|---|---|---|
Dam (GATC) |
5'...TCATGm6A TC...3'3'... AGTAC Tm6AG...5' |
Impaired |
Dam (GATC) |
5'...Gm6A TCATGm6A TC...3'3'... C Tm6AGTAC Tm6AG...5' |
Not determined |
EcoBI (TGA(N)8TGCT) |
5'...TCATGm6A(N)8 TGCT...3'3'... AGTAC T(N)8m6ACGA...5' |
Impaired |
Cleavage efficiency close to the termini of PCR fragments
| bp from the recognition site to fragment end | ||||
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 |
| 20-50 | 50-100 | |||
Number of recognition sites in DNA molecules
| Lambda | ΦX174 | M13mp18/19 | pBR322 | puc18/19 | pUC57 |
|---|---|---|---|---|---|
| 8 | 3 | 1 | 4 | 3 | 3 |
| pTZ19R/U | pTZ57R | pBluescriptIIKS(±) | pBluescriptIISK(±) | pACYC177 | pACYC184 |
| 2 | 2 | 2 | 2 | 4 | 1 |
New sites generated by ligation
Newly generated recognition sites resulting from ligation of protruding compatible DNA ends
| Recognition Sequence | Second Enzyme | Enzymes recognizing newly generated recognition sequence |
|---|---|---|
T^CATGA |
|
|
Newly generated recognition sites resulting from fill-in of 5'-overhang and self-ligation
| Recognition Sequence | Newly generated sequence after reaction | Enzymes that cleave the newly generated sequence |
|---|---|---|
T^CATGA |
TCATGCATGA |
|
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