Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 55°C in a total reaction volume of 50 µl.

Reaction Conditions

1X NEBuffer™ 3.1
Incubate at 55°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

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

Diluent Compatibility

• Diluent B

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

80°C for 20 min

Methylation Sensitivity

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

Activity at Temperature

@37°C: 20%

Companion Products

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

Materials Sold Separately

• Gel Loading Dye, Purple (6X)
• NEBuffer 3.1

1. May exhibit star activity in NEBuffer 2.1.
2. BsmBI is an isoschizomer of Esp3I.

1. Why is there a BsmBI site in LITMUS 38 but not LITMUS 39?
2. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
3. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
4. How can I access the old NEBuffer Activity Chart?
5. My restriction enzyme used to work well in the old NEBuffer but the new Performance chart indicates it has lower activity even though the only difference is the addition of BSA and removal of DTT to the new buffers. Why?
6. Why is my Restriction Enzyme not cutting DNA?
7. Why do I see a DNA smear on an agarose gel after a restriction digest?
8. Why do I see additional DNA bands on my gel after a restriction digest?
9. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
10. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
11. Which restriction enzymes are used in GoldenBraid Assembly?
12. Which restriction enzymes are used in Golden Gate Assembly?

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

• Alphabetized List of Recognition Specificities
• Buffer and Diluent Formulation Table
• Compatible Cohesive Ends and Generation of New Restriction Sites
• Cross Index of Recognition Sequences
• Dam-Dcm and CpG Methylation
• Enzymes with Nonpalindromic Sequences
• Frequencies of Restriction Sites
• Isoschizomers
• Time-Saver™ Qualified Enzymes
• Type IIS Restriction 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
• Effects of CpG Methylation on Restriction Enzyme Cleavage
• Heat Inactivation
• Megabase Mapping
• Restriction Endonucleases - Survival in a Reaction
• Restriction Enzyme Diluent Buffer Compatibility
• Restriction Enzyme Tips
• Single Letter Codes
• Star Activity

• DNA Sequences and Maps Tool

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

• Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M (2014) A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants PLoS One; 9(2), e88218. PubMedID: 24551083 , DOI: 10.1371/journal.pone.0088218

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]

• R0580S_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]

• R0580S_L_v1_0121403
• R0580S_L_v1_0121301
• R0580S_L_v1_0121306
• R0580S_L_v1_0121401
• R0580S_L_v1_0121407
• R0580S_L_v1_0121410
• R0580S_L_v1_0121501
• R0580S_L_v1_0121503
• R0580S_L_v1_0121507
• R0580S_L_v1_0121512
• R0580S_L_v1_0121607
• R0580S_L_v1_0121701
• R0580S_L_v1_0121706
• R0580S_L_v1_0121711

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

• BsmBI
• Gel Loading Dye, Purple (6X)
• NEBuffer™ 3.1

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.