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Restriction Endonucleases >
Restriction Endonucleases >
NmeAIII |
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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:
An E.coli strain that carries the cloned NmeAIII gene from Neisseria meningitidis 2491 (Achtman, M.)
Reagents Supplied:
NEBuffer 4 (10X)
S-adenosylmethionine (SAM) (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 10% | | NEBuffer 2: | | 10% | | NEBuffer 3: | | 0% | | 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.
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 1.00 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 80 μM S-adenosylmethionine (SAM)
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:
2,000 units/ml
Unit Assay Substrate:
ΦX174 RF I DNA
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- Overdigestion with > 5 units of NmeAIII per µg of DNA and incubations > 1 hour are not recommended.
Requires S-adenosylmethionine for optimal activity (supplied with enzyme)
NmeAIII requires two copies of its recognition sequence for cleavage to occur. Thus, the single NmeAIII site in pUC19 is resistant to cleavage. A 10-fold overdigestion cuts less than half of the DNA.
The cleavage point may shift one base pair depending on the DNA sequence context between the recognition site and the position of cleavage. For a given sequence, generally one cut site will predominate.
Significant cleavage occurs on ice and at 25°C.
* NmeAIII produces a stable partial digestion pattern even with excess enzyme. 1 unit is defined as the amount of enzyme required to produce this stable partial digestion pattern.
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 5-fold overdigestion with NmeAIII, > 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, < 5% can be recut with NmeAIII.
Reagents Sold Separately
NEBuffer 4
S-adenosylmethionine (SAM)
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