Nt.AlwI

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.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X

Properties and 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 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

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

Diluent Compatibility

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

80°C for 20 min

Methylation Sensitivity

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

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). Protein Expr. Purif. 69, 226-234. [Epub 2009 Sep 9].

FAQs

  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. 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?
  8. What is the activity of nicking enzymes at different temperatures?

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).

Protocols

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

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Citations

  • Ying Chen, Yun Xiang, Ruo Yuan, Yaqin Chai (2014). A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA Nanoscale. 7(3), 981-6. PubMedID: 25465224, DOI: 10.1039/c4nr05387g

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. Further information regarding NEB product quality can be found here.
  • 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.

Certificate of Analysis

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]

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]

Safety Data Sheet

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