Product Class: Restriction Endonuclease

PI-PspI

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). PI-PspI is a product of in vivo protein splicing that gives rise to both polymerase and endonuclease from a single polypeptide precursor.

Product Source

An E. coli strain that carries the PI-PspI gene from Pyrococcus species (H.W. Jannasch).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer PI-PspI-2010X
pAKR7 XmnI-linearized Control Plasmid500 μg/ml
BSA-20100X

Properties and Usage

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.

Reaction Conditions

1X NEBuffer PI-PspI
Supplement with BSA
Incubate at 65°C

1X NEBuffer PI-PspI:
10 mM Tris-HCl
10 mM MgCl2
1 mM DTT
150 mM KCl
pH 9.2 @ 25°C

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

No

Heat Inactivated

No

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Functional Test (Homing Endonuclease):
    The Homing Endonuclease is incubated with a linearized DNA substrate containing the appropriate cleavage site resulting in the expected sized fragments as determined by agarose gel electrophoresis
  • Ligation and Recutting (Terminal Integrity):
    After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Supporting Documents

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

  1. 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.
  2. Digests of DNA embedded in agarose should be performed with 1 unit of enzyme per µg of DNA for 3 hours at 50°C.
  3. PI-PspI can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis. To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.
  4. Incubation at 37° results in 5% activity.
  5. Supplied with plasmid DNA. XmnI-linearized  pAkR7 is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.7 kb plasmid gives fragments of 2146 and 1532 base pairs.  
  1. How can this enzyme be inactivated?
  2. How can I access the old NEBuffer Activity Chart?
  3. How can I access the old Double Digest Finder?
PI-PspI (or PI-SceI or I-CeuI) can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis.
To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.