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Restriction Endonucleases >
Restriction Endonucleases >
PstI |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the PstI gene from Providencia stuartii 164 (ATCC 49762).
Reagents Supplied:
NEBuffer 3 (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 75% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 50% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
200 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Number of units required to cleave 1 μg of supercoiled plasmid DNA in one hour: pUC19 = 1 unit, pBR322 = 1 unit, LITMUS = 1 unit.
FAQs
- When is star activity a problem for PstI?
- Does spermidine increase activity?
- Are there specific surrounding sequences that produce slow or resistant sites?
- Is PstI inhibited by dUTP incorporated at the site?
- What is the molecular weight of PstI?
- Is PstI blocked by methylation?
- What is the activity of PstI at 25°C?
- Is PstI used in special techniques?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 200-fold overdigestion with PstI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with PstI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 400 units of PstI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 150 units of PstI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Exonuclease Activity:
Incubation of 400 units of PstI with 1 μg of sonicated 3H DNA (105 cpm/μg) for 4 hours at 37ºC in 50 μl reaction buffer released < 0.1% radioactivity.
Reagents Sold Separately
NEBuffer 3
BSA
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