NotI-HF® RE-Mix® is a ready to use 10X master mix combining the restriction enzyme, NotI-HF (NEB #R3189 ), NEBuffer, BSA and loading dye in a single tube. Simply add DNA and water and incubate for 15 minutes to perform the restriction digest.
Product SourceAn E. coli strain that carries the cloned and modified NotI gene from Nocardia otitidiscaviarum (ATCC 14630)
Reaction Definition1X NotI-HF RE-Mix and DNA in a total reaction volume of 20 μl. Incubate at 37°C for 15 minutes.
One reaction is defined as the amount of enzyme that will digest 1 μg of DNA in 15 minutes at 37°C in a total reaction volume of 20 μl.
Properties and Usage
Heat Inactivation65°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked
Quality Control AssaysThe 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.
- Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
- 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.
Relevant patent applications include: US2009-0029376, EP2390316, CN101802183A, IN 7681/CHENP/2009 and JP 2010-516300.
- What are the advantages of using a RE-Mix Restriction Enzyme Master Mix?
- For some restriction enzymes star activity can be a concern. Is this also applicable to the RE-Mix?
- The bands on my gel are not as sharp as I would like. Is there a way to improve the sharpness?
- Is it possible to use RE-Mix in double digests?
- Can RE-Mix be used in overnight digestions?
- Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
Usage Guidelines & Tips
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Activity of Restriction Enzymes in a Q5®, Taq or Phusion PCR Mix
- Alteration of Apparent Recognition Specificities Using Methylases
- Cleavage Close to the End of DNA Fragments
- 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
- Optimizing Restriction Endonuclease Reactions When Using RE-Mix® Restriction Enzyme Master Mix
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- Single Letter Codes
- Star Activity
- Traditional Cloning Quick Guide