Equipamentos para Laboratórios | Equipamento para Laboratório

Recognition Site:

Source:
A 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:

1. Less than 50% of AflII fragments ligate in a standard 20 μl reaction containing 100-500 units of T4 DNA Ligase.
2. AflII is inhibited by salt concentrations > 50 mM.

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 (205 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

Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,030,569

AflII FAQ

Q1: What factors inhibit AflII?
A1: AflII is inhibited by salt concentrations greater then 50 mM. Some mini prep DNA may contain residual salt.  A 70% ethanol wash or dialysis is recommended to remove the salt.

Q2: 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?
A2: All our restriction enzymes were re-assayed in both NEBuffer 4 vs. the previously supplied NEBuffer. In a few cases minor formulation changes were required to bring the activity to 100% in NEBuffer 4.

Q3: Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
A3: In general the properties of the restriction enzyme remain the same although for some enzymes, some minor changes in heat inactivation, Time-Saver™ qualification, etc. were observed. All the updated information can be found on the supplied data card as well as at www.neb.com.

Q4: If I have an old tube of enzyme, what NEBuffer should I use?
A4: Our restriction enzymes are color coded on the label for the appropriate NEBuffer and can either be used with the previously supplied NEBuffer or with NEBuffer 4. To ensure 100% cleavage in NEBuffer 4, additional units of enzyme and/or longer incubation time may be necessary.

Q5: Will the new enzyme work in the originally supplied NEBuffer?
A5: Yes it will. In all instances the restriction enzyme will have 100% activity in the originally supplied NEBuffer.

Q6: Why is NEB switching this restriction enzyme to NEBuffer 4?
A6: Our main goal is to simplify the NEBuffer system so that the majority of restriction enzymes are compatible in a single buffer. We now supply 162 restriction enzymes with NEBuffer 4 including all the new High Fidelity (HF) restriction enzymes.

Q7: Why don't AflII- generated DNA ends ligate well?
A7: We don't know. There is something odd about the DNA conformation of these ends.

Q8: How can ligation efficiency be increased?
A8: Less than 50% of AflII fragments ligate in a standard 20 microliter reaction containing 100-500 units of T4 DNA ligase. Ligation can be enhanced by using 1 microliter of high concentration T4 DNA ligase. Blunting the ends, wither by fill-in or removal, increases ligation of the poorly- ligated ends.

Q9: What is the molecular weight of AflII?
A9: The MW of AflII is 36,778 Da from sequence data.

Q10: What is the activity of AflII at 25°C?
A10: AflII is 25-50% active at 25°C.