DescriptionThe intron encoding I-CeuI is present in the chloroplast large rRNA gene of Chlamydomonas eugametos (1). This gene has been cloned and overexpressed in E. coli (2).
Product SourceAn E. coli strain that carries the I-CeuI gene from Chlamydomonas eugametos (C. Lemieux).
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|pBHS ScaI-linearized Control Plasmid||500 μg/ml|
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to cleave 1 µg of pBHS ScaI-linearized Control Plasmid in 3 hours at 37°C in a total reaction volume of 50 µl.
1X CutSmart® Buffer
Incubate at 37°C
1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C
Activity in NEBuffersNEBuffer 1.1: 10%
NEBuffer 2.1: 10%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 μg/ml BSA
pH 7.4 @ 25°C
Heat Inactivation65°C for 20 min
Quality Control AssaysThe 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.
- 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.
- 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.
- Supplied with plasmid DNA. ScaI-linearized pBHS is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.5 kb plasmid gives fragments of 2100 and 1400 base pairs.
- Can a double digest be performed with PI-SceI and I-CeuI?
- What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
- How can I access the old NEBuffer Activity Chart?
- How can I access the old Double Digest Finder?
- I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason?