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Restriction Endonucleases >
Restriction Endonucleases >
NlaIII |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the NlaIII gene from Neisseria lactamica (NRCC 2118).
Reagents Supplied:
NEBuffer 4
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 25% | | 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:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
65°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 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 ΦX174 RF I DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
ΦX174 DNA
Storage Conditions:
10 mM Tris-HCl
200 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
For long term storage (>30 days), store at -70°C.
Diluent Compatibility:
Diluent B
Notes
General notes:- Cleaves to leave a 3´ CATG extension which can be ligated to DNA fragments generated by SphI.
- The half life of NlaIII is 6 months at -70°C.
Usage notes:- NaCI and KCI inhibit activity, (NH4)2 SO4 does not.
FAQs
- Is NlaIII inhibited by salt?
- Are storage conditions for NlaIII other than -20°C recommended?
- Is activity loss in NlaIII seen in 6 months or less?
- Does NlaIII produce commonly used compatible ends?
- Is extended digestion of NlaIII recommended?
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 15-fold overdigestion with NlaIII, > 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 NlaIII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 75 units of NlaIII 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 NlaIII with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.15% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,278,060
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