Having supplied restriction enzymes to the research community for over 40 years, NEB has earned the reputation of being the leader in enzyme technologies. Working continuously to be worth of that distinction, NEB strives to develop enzyme of the highest purity and unparalleled quality.
Reduce Star Activity with High-Fidelity Restriction Enzymes
Standard Protocol for Restriction Enzyme Digests
NEB TV Episode 15
CutSmart Restriction Enzyme Buffer
Restriction Enzyme Digest Protocol: Cutting Close to DNA End
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel
Why is My Restriction Enzyme Not Cutting DNA?
Restriction Enzyme Digest Problem: Too Many DNA Bands
Double Digestion with NEBcloner
Product SourceAn E. coli strain that carries the AlwI gene from Acinetobacter lwoffii (R. Morgan).
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|CutSmart® Buffer||-20||10 X|
- Product Categories:
- Restriction Endonucleases: A
- Restriction Enzyme Digestion
Properties and Usage
Unit DefinitionOne 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.
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 NEBuffersCutSmart® Buffer: 100%
NEBuffer 1.1: 50%
NEBuffer 2.1: 50%
NEBuffer 3.1: 10%
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
pH 7.4 @ 25°C
dam methylation: Blocked
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive
- 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.
- This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
- AlwI produces DNA fragments that have a single-base 5´ extension which are more difficult to ligate than blunt-ended fragments.
Faqs & Tech Tips
- Is AlwI blocked by dam methylation?
- What is the activity of AlwI at 25°C?
- Why is AlwI- cut DNA difficult to ligate?
- Why is an extended digest not recommended?
- How can I increase ligation efficiency?
- 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?
- My enzyme is no longer Time-Saver™ qualified. What happened?
- What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
- How can I access the old NEBuffer Activity Chart?
- 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?
- 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?
- Why is my Restriction Enzyme not cutting DNA?
- Why do I see a DNA smear on an agarose gel after a restriction digest?
- Why do I see additional DNA bands on my gel after a restriction digest?
- How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
This enzyme produces a single base extension, which is difficult to ligate.
Protocols & Manuals
Other Tools & Resources
- 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
- Time-Saver™ Qualified Enzymes
- Type IIS Restriction Enzymes
- Why Choose Recombinant Enzymes?
Usage Guildelines & Tips
- 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
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- Restriction Enzyme Tips
- Single Letter Codes
- Star Activity
- Traditional Cloning Quick Guide
Troubleshooting GuidesRestriction Enzyme Troubleshooting Guide
Quality & Safety
Quality Assurance StatementQuality 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.
SpecificationsThe 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 AnalysisThe 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]
Safety DataSheetsThe following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
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 email@example.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.