Product Class: Restriction Endonuclease

Nt.AlwI
neb31 cloned at NEB recombinant dil_A 37 80 Heat dam

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

NEB restriction endonuclease that recognizes the sequence GGATCNNNN^N
Isoschizomers | Single Letter Code

Product Introduction

Catalog # Size Concentration
R0627S 500.0 units 10000 units/ml

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Product Information

Description

Nt.AlwI is a nicking endonuclease that cleaves only one strand of DNA on a double-stranded DNA substrate. It is an engineered derivative of AlwI which catalyzes a single strand break 4 bases beyond the 3´ end of the recognition sequence on the top strand.

Product Source

An E. coli strain containing a chimeric gene encoding the DNA recognition domain of AlwI and the cleavage/dimerization domain of Nt.BstNBI.
This product is related to the following categories:
Nicking Endonucleases Products,
Restriction Endonucleases: N-O Products,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to convert 1 µg of supercoiled pUC101 DNA (dam-/dcm-) to open circular form in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X rCutSmart™ Buffer
Incubate at 37°C

1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Activity in NEBuffers

NEBuffer™ r1.1: 10%
NEBuffer™ r2.1: 100%
NEBuffer™ r3.1: 100%
rCutSmart™ Buffer: 100%

Diluent Compatibility

Storage Buffer

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

80°C for 20 minutes

Methylation Sensitivity

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

Product Notes

  1. This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
  2. To see how this enzyme (along with other nicking enzymes) performs at different temperatures, please visit Effect of Various Temperatures of Different Nicking Endonucleases.

References

  1. Song, Q. et al. (2010). Anal.Chem. [Epub ahead of print].
  2. Zhang, P. et al. (2010). ProteinExpr. Purif. 69, 226-234. [Epub 2009 Sep 9].

Protocols, Manuals & Usage

Protocols

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

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the activity of nicking enzymes at different temperatures?
  2. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?
  3. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

Troubleshooting

Tech Tips

This enzyme will not cut plasmids isolated from most E.coli because it is blocked by dam methylation. For enzyme cleavage to occur, plasmids must be transformed and isolated from a dam-, dcm- strain of E.coli, such as NEB’s dam-/dcm- Competent E.coli (NEB #C2925).

Quality, Safety & Legal

Quality Assurance Statement

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.

Specifications

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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