Unit Definition

One unit is defined as the amount of enzyme required to cleave 1 µg of pGPS2 NotI-Iinearized Control Plasmid in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

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 NEBuffers

CutSmart® Buffer: 100%
NEBuffer 1.1: 10%
NEBuffer 2.1: 50%
NEBuffer 3.1: 25%

Diluent Compatibility

• Diluent B

Storage Temperature

-80°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

65°C for 20 min

Companion Products

• Monarch® Plasmid Miniprep Kit 
• Monarch® DNA Gel Extraction Kit
• Monarch® PCR & DNA Cleanup Kit (5 μg)

1. The enzyme retains full activity after at least 10 free-thaw cycles; the enzyme can be aliquoted and stored at –80C.
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. Supplied with plasmid DNA. NotI-linearized pGPS2 is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 2499 bp plasmid with I-SceI gives fragments of 1518 and 981 base pairs.

1. My enzyme used to come with another NEBuffer, but CutSmart™ Buffer is now recommended?  Why?
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. 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?
5. Why is my Restriction Enzyme not cutting DNA?
6. Why do I see a DNA smear on an agarose gel after a restriction digest?
7. Why do I see additional DNA bands on my gel after a restriction digest?
8. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

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

• Alphabetized List of Recognition Specificities
• Buffer and Diluent Formulation Table
• Cross Index of Recognition Sequences
• Dam-Dcm and CpG Methylation
• Enzymes with Nonpalindromic Sequences
• Frequencies of Restriction Sites
• Isoschizomers
• Why Choose Recombinant Enzymes?

• Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
• Activity of Restriction Enzymes in PCR Buffers
• Cleavage Close to the End of DNA Fragments
• Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
• Double Digests
• Heat Inactivation
• Megabase Mapping
• NEBuffer Activity/Performance Chart with Restriction Enzymes
• Optimizing Restriction Endonuclease Reactions
• Restriction Endonucleases - Survival in a Reaction
• Restriction Enzyme Diluent Buffer Compatibility
• Restriction Enzyme Tips
• Single Letter Codes
• Star Activity
• Traditional Cloning Quick Guide

• Restriction Enzyme Troubleshooting Guide

• DNA Sequences and Maps Tool

• Double Digest Finder
• Enzyme Finder
• NEBcutter V2.0
• NEBioCalculator
• REBASE

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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]

• R0694S_L_v1

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]

• R0694S_L_v1_0121405
• R0694S_L_v1_0111302
• R0694S_L_v1_0111306
• R0694S_L_v1_0111310
• R0694S_L_v1_0111312
• R0694S_L_v1_0121411
• R0694S_L_v1_0121504
• R0694S_L_v1_0131504
• R0694S_L_v1_0131509
• R0694S_L_v1_0131605
• R0694S_L_v1_0141605
• R0694S_L_v1_0201804

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

• I-SceI
• CutSmart® Buffer
• pGPS2 NotI-linearized Control Plasmid

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.