Product Class: Restriction Endonuclease

AlwI

cloned at neb recombinant icon_CutSmart incubation temp heat inactivation no dam
Alw-I-cutsite_1
Catalog #SizeConcentration
R0513S500 units10,000 units/ml
R0513L2,500 units10,000 units/ml

Description

Product Source

An E. coli strain that carries the AlwI gene from Acinetobacter lwoffii (R. Morgan).

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 digest 1 µg of λ DNA (dam-) in 1 hour at 37°C in 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: 50%
NEBuffer 2.1: 50%
NEBuffer 3.1: 10%
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

No

Methylation Sensitivity

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

Heat Inactivated

No

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.
  • Ligation and Recutting (Terminal Integrity):
    After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
  • 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.

Supporting Documents

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

Notes

  1. AlwI produces DNA fragments that have a single-base 5´ extension which are more difficult to ligate than blunt-ended fragments. 
  2. This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
  3. For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.
  4. Star activity may result from extended digestion, high enzyme concentration or a glycerol concentration of >5%
  1. Is AlwI blocked by dam methylation?
  2. What is the activity of AlwI at 25°C?
  3. Why is AlwI- cut DNA difficult to ligate?
  4. Why is an extended digest not recommended?
  5. How can I increase ligation efficiency?
  6. My restriction enzyme used to be available at a lower concentration. Why does it now come at a higher concentration of 10,000 u/ml?
  7. My enzyme is no longer Time-Saver™ qualified. What happened?
  8. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  9. How can I access the old NEBuffer Activity Chart?
  10. 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?
  11. 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?
  12. Why is my Restriction Enzyme not cutting DNA?
  13. Why do I see a DNA smear on an agarose gel after a restriction digest?
  14. Why do I see additional DNA bands on my gel after a restriction digest?
  15. 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

This enzyme produces a single base extension, which is difficult to ligate.