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Restriction Endonucleases >
Nicking Endonucleases >
Nt.AlwI |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | single letter code
Description:
Nt.AlwI is a nicking endonuclease that cleaves only one strand of DNA on a double-stranded DNA substrate. It is an engineered derivative of AlwI which catalyzes a single strand break 4 bases beyond the 3´ end of the recognition sequence on the top strand.
Source:
An E. coli strain containing a chimeric gene encoding the DNA recognition domain of AlwI and the cleavage/dimerization domain of Nt.BstNBI.
Reagents Supplied:
NEBuffer 2
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 25% | | NEBuffer 4: | | 50% |
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: Blocked | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
80°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 2
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 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 convert 1 µg of supercoiled pUC101 DNA (dam-/dcm-) to open circular form in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
pUC101 DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of Nt.AlwI 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 50 units of Nt.AlwI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.05% of the total radioactivity.
Reagents Sold Separately
NEBuffer 2
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 6,395,523
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