Product SourceAn E. coli strain that carries the cloned EcoP15 l res-mod genes from plasmid pSHl180 (D.N. Rao)
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
Properties and Usage
Unit DefinitionOne unit is defined as the amount enzyme required to digest 1 µg of pUC19 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
1X NEBuffer 3.1
Supplement with ATP
Incubate at 37°C
1X NEBuffer 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C
Activity in NEBuffersNEBuffer 1.1: 75%
NEBuffer 2.1: 100%
NEBuffer 3.1: 100%
CutSmart® Buffer: 100%
10 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
pH 7.4 @ 25°C
Heat Inactivation65°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive
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.
- 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.
- ATP is required for DNA cleavage. Efficient cleavage requires the presence of two inversely oriented recognition sites. A head to head orientation is preferred. The "head" of the sequence is defined as the dG at the 3´ end of the CAGCAG strand (top strand). Cleavage efficiency is also affected by the distance between the two sites.
- Not sensitive to dam, dcm or mammalian CpG methylation.
- EcoP15I requires two copies of its recognition sequence for cleavage to occur, with limited cleavage on plasmids with a single site.
- Elisabeth Möncke-Buchner, Maja Rothenberg, Stefanie Reich, Katja Wagenführ, Hideo Matsumura, Ryohei Terauchi, Detlev H. Krüger, and Monika Reuter (2009). Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target. J. Mol. Biol. 387, 1309–1319.
- What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
- Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
- How can I access the old NEBuffer Activity Chart?
- Are there any tips to get optimal cutting with restriction enzyme EcoP15I?