Product Class: Restriction Endonuclease


Did you know this product can be customized or purchased in larger volumes? Submit an inquiry to find out more.
The new and current Double Digest Finder and current Activity/Performance Chart for the CutSmart buffer system are available. The previous version of the Double Digest Finder, as well as the previous Version of Activity/Performance Chart that use the former buffer system, are still available for your convenience.

recombinant timesaver 5min incubation temp heat inactivation
Catalog #SizeConcentration
R0646S500 units10,000 units/ml
R0646L2,500 units10,000 units/ml


Product Source

An E. coli strain that carries the cloned EcoP15 l res-mod genes from plasmid pSHl180 (D.N. Rao)

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
10X ATP10X
NEBuffer 3.1-2010X

Properties and Usage

Unit Definition

One 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.

Reaction Conditions

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 NEBuffers

NEBuffer 1.1: 75%
NEBuffer 2.1: 100%
NEBuffer 3.1: 100%
CutSmart™ Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

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

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Heat Inactivated


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.
  • 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

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]
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]
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
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 or fill out the Technical Support Form for appropriate document.


  1. 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.
  2. Not sensitive to dam, dcm or mammalian CpG methylation.
  3. EcoP15I requires two copies of its recognition sequence for cleavage to occur, with limited cleavage on plasmids with a single site.


  1. 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.
  1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  2. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  3. How can I access the old NEBuffer Activity Chart?
  4. How can I access the old Double Digest Finder?
  5. Are there any tips to get optimal cutting with restriction enzyme EcoP15I?
  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes

Selection Tools

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Application Notes


  • Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. (2013). Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research. DOI: doi:10.1093/nar/gkt601