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| | | | NotI | | | NotI has a High Fidelity (HF) Version available | Receive a free DNA Ladder Sample with the purchase of this enzyme through 9/30/2010! | |
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R0189S |
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500 units
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10,000 units/ml |
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R0189L |
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2,500 units
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10,000 units/ml |
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R0189M |
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2,500 units
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50,000 units/ml |
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Recognition Site:
| single letter code
Description:
NotI has a High Fidelity version NotI-HF™ (NEB #R3189).
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity and have 100% activity in buffer 4 which may simplify your double digest.
Source:
A E. coli strain that carries the NotI gene from Nocardia otitidis-caviarum (ATCC 14630).
Reagents Supplied:
NEBuffer 3 (10X)
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 25% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Blocked | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ +) Intermediate activity. Suitable for extended digestion, but < 8 hours.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
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:
10,000 units/ml and 50,000 units/ml
Unit Assay Substrate:
pBC4 DNA
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Supercoiled plasmids may require up to 5-fold more NotI for complete digestion than linear DNAs.
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, > 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.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 200 units of NotI 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 with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 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 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.
Reagents Sold Separately
NEBuffer 3
BSA
Companion Products
NotI-HF™
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,371,006
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