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R0520L |
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10,000 units
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20,000 units/ml |
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R0520S |
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2,000 units
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20,000 units/ml |
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Recognition Site:
| single letter code
Source:
An E. coli strain that carries the AflII gene from Anabaena flos-aquae (CCAP 1403/13f).
Reagents Supplied:
NEBuffer 4 (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 100% | | 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 | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes
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 RFI DNA in 1 hour at 37°C in a total reaction volume of 50 μl.
Concentration:
20,000 units/ml
Unit Assay Substrate:
ΦX174 DNA
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.5 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- Less than 50% of AflII fragments ligate in a standard 20 μl reaction containing 100-500 units of T4 DNA Ligase.
- AflII is inhibited by salt concentrations > 50 mM.
FAQs
- What factors inhibit AflII?
- The NEB catalog has historically stated that activity in NEBuffer 4 is less than 100% yet this enzyme is now supplied with NEBuffer 4. What have you changed?
- Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
- If I have an old tube of enzyme, what NEBuffer should I use?
- Will the new enzyme work in the originally supplied NEBuffer?
- Why is NEB switching this restriction enzyme to NEBuffer 4?
- Why don't AflII- generated DNA ends ligate well?
- How can ligation efficiency be increased?
- What is the molecular weight of AflII?
- What is the activity of AflII at 25°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 5-fold overdigestion with AflII, approximately 50%
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 AflII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 300 units of AflII 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 AflII with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.4% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of AflII with 1 μg of
pBR322 DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Reagents Sold Separately
NEBuffer 4
BSA
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