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Restriction Endonucleases >
Homing Endonucleases >
PI-PspI |
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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
Description:
PI-PspI is obtained from a strain of E. coli which expresses the DNA polymerase from the extreme thermophile, Pyrococcus species GB-D (1). The endonuclease is a product of in vivo protein splicing that gives rise to both polymerase and endonuclease from a single polypeptide precursor.
Source:
A E. coli strain that carries the PI-PspI gene from Pyrococcus species (H.W. Jannasch).
Reagents Supplied:
NEBuffer PI-PspI (10X)
BSA (100X)
pAKR7 XmnI-linearized Control Plasmid (5 μg)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 10% | | NEBuffer 3: | | 10% | | NEBuffer 4: | | 10% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Activity at 37°C:
5%
Heat Inactivation:
No
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer PI-PspI
Supplemented with 100 μg/ml Bovine Serum Albumin and 5 μg pAKR7 XmnI-linearized Control Plasmid
Incubate at
65°C.
1X NEBuffer PI-PspI:
10 mM Tris-HCl
150 mM KCl
10 mM MgCl2
1 mM Dithiothreitol
pH 9.2 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to cleave 1 µg of pAKR7 XmnI-linearized Control Plasmid in 1 hour at 65°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
pAKR7 XmnI-linearized Control Plasmid
Storage Conditions:
10 mM Tris-HCl
200 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
Homing endonucleases do not have stringently-defined recognition sequences in the way that restriction enzymes do. That is, single base changes do not abolish cleavage but reduce its efficiency to variable extents. The precise boundary of required bases is generally not known. The recognition sequence listed is one site that is known to be recognized and cleaved.
Double-stranded cleavage at the site indicated by arrows yields a four base, 3´ extension. The sequence degeneracy tolerated by this enzyme has not yet been determined. However, digestion patterns from bacterial and yeast chromosomal DNAs indicate that the observed sequence specificity is 8–10 bases under stringently controlled conditions.General notes:- Digests should be incubated in 0.5% SDS prior to electrophoresis. Digests of DNA embedded in agarose should be performed with 1 unit of enzyme per µg of DNA for 3 hours at 50°C.
Incubation at 37° results in 5% activity.
FAQs
- How can this enzyme be inactivated?
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 5-fold overdigestion with PI-PspI, > 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 PI-PspI.
Cleavage by PI-PspI leaves DNA fragments with four nucleotide 3' extensions. Fragments with complimentary ends can be joined by T4 DNA Ligase.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 5 units of PI-PspI incubated for 16 hours at 65ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of PI-PspI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 65ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of PI-PspI with 1 μg of
ΦX174 RF I DNA for 4 hours at 65ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Plasmid DNA:
pAKR7 XmnI-linearized Control Plasmid is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Cleavage of this 3.7 kb plasmid gives fragments of 2,146 and 1,532 base pairs.
Reagents Sold Separately
NEBuffer PI-PspI
BSA
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