PI-SceI

Description

The intein encoding PI-SceI is present in the VMA ATPase gene Saccharomyces cerevisiae (1,5). The gene has been modified for independent expression in E. coli using a T7 RNA polymerase expression system (2).

Product Source

An E. coli strain that carries the VMA1 ATPase gene from Saccharomyces cerevisiae (J. Thorner).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
pBSvdeX XmnI-linearized Control Plasmid500 μg/ml
Purified BSA-2010 mg/ml
NEBuffer PI-SceI-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave 1 μg of pBSvdeX XmnI-linearized Control Plasmid in 3 hours at 37°C in a total reaction volume of 50 μl.

Reaction Conditions

1X NEBuffer PI-SceI
Supplement with Purified BSA
Incubate at 37°C

1X NEBuffer PI-SceI:
10 mM MgCl2
1 mM DTT
10 mM Tris-HCl
100 mM KCl
pH 8.6 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 10%
NEBuffer 2.1: 10%
NEBuffer 3.1: 10%
CutSmart® Buffer: 10%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

65°C for 20 min

Notes

  1. Supplied with plasmid DNA: XmnI-linearized pBSvdeX is supplied 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.7 kb plasmid with PI-SceI gives fragments of 2550 and 1150 base pairs Enzyme Properties
  2. 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.
  3. PI-SceI 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.

FAQs

  1. Can a double digest be performed with PI-SceI and I-CeuI?
  2. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  3. How can I access the old NEBuffer Activity Chart?
  4. Why is my Restriction Enzyme not cutting DNA?
  5. Why do I see a DNA smear on an agarose gel after a restriction digest?
  6. Why do I see additional DNA bands on my gel after a restriction digest?
  7. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Tech Tips

PI-SceI 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.

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

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. Further information regarding NEB product quality can be found here.
  • 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.

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]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.