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PstI | Enzimas "P" Cellco
PstI
Ficha técnica
| Código | PstI |
| Categoria | Enzimas de Restrição |
| Marca | Cellco |
Descrição Geral
PstI | Enzimas "P" Cellco
Isoschizomers: BspMAI
For general laboratory use.
Unit Definition:
One unit is the amount of enzyme required to completely digest 1 μg of Lambda DNA (28 sites) in 1 hour in a total reaction volume of 50 μl. Enzyme activity was determined in the recommended reaction buffer.
Source:
Providencia stuartii, recombinant, E. coli
Providencia stuartii, recombinant, E. coli
Shipping:
shipped on gel packs
Storage Conditions:
store at -20 °C
avoid freeze/thaw cycles
Shelf Life:
12 months
Form:
liquid (Supplied in 10 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA, 0.15 % Triton X-100 and 50 % [v/v] glycerol)
Concentration:
10 units/μl
Supplied with:
10x Universal Buffer (UB)
Recommended 50 μl assay
| Components | 50 µL rxn |
| 10X Universal Buffer (UB) | 5 µL |
| pure DNA1 or PCR product2 | 1 μg |
| enzyme | 5 units |
| PCR grade water | fill up to 50 μl |
1 Supercoiled or high molecular weight DNA (e.g. plant genomic DNA) may require longer incubation time or higher amount of enzyme.
2 Some enzymes may require additional DNA bases flanking the restriction site for complete digestion.
Double Digestion - Buffer Compatibility
B1 - 10-25% Relative Activity
B2 - 50-75% Relative Activity
B3 - 75-100% Relative Activity
B4 - 50-75% Relative Activity
B5 - 50% Relative Activity
1x UB - 100% Relative Activity (recommended)
B1 - 10-25% Relative Activity
B2 - 50-75% Relative Activity
B3 - 75-100% Relative Activity
B4 - 50-75% Relative Activity
B5 - 50% Relative Activity
1x UB - 100% Relative Activity (recommended)
Please note that the optimum digestion condition for this enzyme is 1x UB. Within the Universal Buffer (UB) system, the most majority of our enzymes display 100% Relative Activity in 1x UB and only few either in 0.5x or 2x UB. If optimum condition for second enzyme is different than the recommended for the first enzyme, we suggest carrying out first the restriction at the higher recommended concentration of UB and dilute the reaction volume to the adequate UB concentration for further proceeding with the second restriction.
Protocol:
- The enzyme should not exceed 10 % of total reaction volume.
- Add enzyme as last component. Mix components well before adding enzyme. After enzyme addition, mix gently by pipetting. Do not vortex.
- Incubate 60 min. at 37 °C.
- Stop reaction by alternatively:
- Addition of 2.1 μl EDTA pH 8.0 [0.5 M], final 20 mM
- Heat Inactivation (15 min. at 55 °C)
- Spin Column DNA Purification (e.g. PCR Purification Kit, Cat. No. PP-201S/L)
- Gel Electrophoresis and Single Band Excision (e.g. Agarose Gel Extraction Kit, Cat.-No. PP-202S/L)
- Phenol-Chloroform Extraction or Ethanol Precipitation.
Reaction Enzymes Buffer Guide:
| Buffer 1 | 10 × B1 | 100 mM 100 mM 1000 μg/ml | Tris-HCl (pH 7.9, 25°C) MgCl2 BSA |
| Buffer 2 | 10 × B2 | 100 mM 100 mM 500 mM 1000 μg/ml | Tris-HCl (pH 7.9, 25°C) MgCl2 NaCl BSA |
| Buffer 3 | 10 × B3 | 500 mM 100 mM 1000 mM 1000 μg/ml | Tris-HCl (pH 7.9, 25°C) MgCl2 NaCl BSA |
| Buffer 4 | 10 × B4 | 100 mM 100 mM 1500 mM 1000 μg/ml | Tris-HCl (pH 7.9, 25°C) MgCl2 NaCl BSA |
| Buffer 5 | 10 × B5 | 200 mM 100 mM 500 mM 1000 μg/ml | Tris-acetate (pH 7.9, 25°C) Mg-acetate K-acetate BSA |
Reaction Buffer Compatibility:
Our restriction enzymes are fully compatible to restrictases and buffer systems from other manufacturers and can be used along in double digestions. To obtain best results, consult the corresponding manuals of all involved products.
Ligation and recutting:
After 10-fold overdigestion with PstI, >95% of the DNA fragments can be ligated and recut with this enzyme.
Star activity:
Conditions of high enzyme concentration or glycerol concentration >12% may result in star activity.
Conditions of high enzyme concentration or glycerol concentration >12% may result in star activity.
DNA Methylation:
No Inhibition: dam, dcm, CpG
No Inhibition: dam, dcm, CpG
Quality Control:
All preparations are assayed for contaminating endonuclease, 3’-exonuclease, 5’-exonuclease/ 5’-phosphatase, as well as nonspecific single- and doublestranded DNase activities.
All preparations are assayed for contaminating endonuclease, 3’-exonuclease, 5’-exonuclease/ 5’-phosphatase, as well as nonspecific single- and doublestranded DNase activities.
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