Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 60°C in a total reaction volume of 50 µl.

Reaction Conditions

1X NEBuffer™ 3.1
Incubate at 60°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: 75%
NEBuffer 1.1: 10%
NEBuffer 2.1: 75%
NEBuffer 3.1: 100%

Diluent Compatibility

• Diluent A

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

No

Methylation Sensitivity

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

Activity at Temperature

@37°C: 50%

Companion Products

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

Materials Sold Separately

• NEBuffer 3.1

1. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.
2. Of the over 3,000 known restriction endonucleases, BstEII is one of the few that produces extensions of more than 4 bases.
3. May exhibit Star Activity in NEBuffer 2.1 and CutSmart Buffer.
4. Star activity may result from a glycerol concentration of > 5%.

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. Why is my Restriction Enzyme not cutting DNA?
5. Why do I see a DNA smear on an agarose gel after a restriction digest?
6. Why do I see additional DNA bands on my gel after a restriction digest?
7. 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
• Compatible Cohesive Ends and Generation of New Restriction Sites
• Cross Index of Recognition Sequences
• Dam-Dcm and CpG Methylation
• Enzymes with Multiple Recognition Sequences
• Frequencies of Restriction Sites
• Interrupted Palindromes
• Isoschizomers
• Time-Saver™ Qualified Enzymes
• 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
• Dam and Dcm Methylases of E. coli
• Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
• Double Digests
• Effects of CpG Methylation on Restriction Enzyme Cleavage
• Heat Inactivation
• Megabase Mapping
• NEBuffer Activity/Performance Chart with Restriction Enzymes
• Optimizing Restriction Endonuclease Reactions
• Reduced Star Activities of HF® Enzymes
• 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]

• R0162S_L_v1
• R0162T_M_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]

• R0162S_L_v1_0851309
• R0162S_L_v1_0851405
• R0162S_L_v1_0841212
• R0162S_L_v1_0841304
• R0162T_M_v1_0841212
• R0162T_M_v1_0841304
• R0162T_M_v1_0851405
• R0162T_M_v1_0851410
• R0162S_L_v1_0851410
• R0162S_L_v1_0851504
• R0162T_M_v1_0851504
• R0162T_M_v1_0851509
• R0162S_L_v1_0851510
• R0162S_L_v1_0851604
• R0162T_M_v1_0851606
• R0162S_L_v1_0851610
• R0162T_M_v1_0851703
• R0162S_L_v1_0851709
• R0162T_M_v1_0851712
• R0162S_L_v1_0851804

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

• BstEII
• 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.