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Restriction Endonucleases >
Nicking Endonucleases >
Nt.BstNBI |
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- N.BstNB I catalyzes a single-stranded nick 3' of its recognition sequence.
Recognition Site:
isoschizomers | single letter code
Description:
Nt.BstNBI is a site specific endonuclease that cleaves only one strand of DNA on a double-stranded DNA substrate. The nicking endonuclease catalyzes a single strand break 4 bases beyond the 3´ side of the recognition sequence.
Source:
An E. coli strain that carries the cloned Nt.BstNBI gene from Bacillus stereothermophilus 33M (Z. Chen).
Reagents Supplied:
NEBuffer 3
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 10% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 10% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Activity at 37°C:
10%
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Incubate at
55°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 T7 DNA in 1 hour at 55°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
T7 DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
Usage notes:- 10 units of enzyme are required to convert 1 µg of supercoiled pBR322 or pUC19 DNA to open circular form in 1 hour at 55°C.
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 Nt.BstNBI, > 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 Nt.BstNBI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 60 unit of Nt.BstNBI incubated for 16 hours at 55ºC
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 60 units of Nt.BstNBI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 55ºC
released < 0.1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 3
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 6,191,267
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