Source: An E. coli strain that carries the cloned and modified (K150A) NotI gene from Nocardia otitidis-caviarum (ATCC 14630)
Reagents Supplied: NEBuffer 4 (10X)
BSA (100X)
Enzyme Properties Activity in NEBuffers:
NEBuffer 1:
25%
NEBuffer 2:
100%
NEBuffer 3:
25%
NEBuffer 4:
100%
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
CpG methylation: Blocked
Heat Inactivation: 65°C for 20 minutes
Survival in a Reaction: Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Supplemented with 100 μg/ml Bovine Serum Albumin Incubate at
37°C.
1X NEBuffer 4: 20 mM Tris-acetate 50 mM potassium acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition: One unit is defined as the amount of enzyme required to digest 1 μg of pBC4 DNA in 1 hour at 37°C in a total reaction volume of 50 μl.
Concentration: 20,000 units/ml
Unit Assay Substrate: pBC4 DNA
Storage Conditions: 10 mM Tris-HCl 50 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol
Storage Temperature: -20°C
Diluent Compatibility: Diluent A
Notes General notes:
Supercoiled plasmids may require up to 5-fold more NotI-HF for complete digestion than linear DNAs.
Usage notes:
NotI-HF™ has the same specificity of NotI (NEB #R0189), but it has been engineered for reduced star activity.
Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 10-fold overdigestion with NotI-HF™, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with NotI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 200 units of NotI-HF™ incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of NotI-HF™ with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of NotI-HF™ with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
