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Restriction Endonucleases >
Restriction Endonucleases >
AscI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the AscI gene from Arthrobacter species (R. Morgan).
Reagents Supplied:
NEBuffer 4
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 10% | | NEBuffer 3: | | 10% | | 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: Blocked |
Heat Inactivation:
65°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 4
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 λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
λ 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
Notes
General notes:- AscI is an octanucleotide-recognizing restriction endonuclease pNEB193, a cloning vector which contains single sites for NEB's 8-base cutters AscI, PacI and PmeI is available (NEB #N3051S).
- AscI is strongly inhibited by NaCl and ammonium acetate. Therefore, we recommend using sodium acetate or potassium acetate as the salt for alcohol precipitation of DNA to be cut with AscI.
FAQs
- What factors inhibit AscI?
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 10-fold overdigestion with AscI, > 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 AscI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 30 units of AscI 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 100 units of AscI 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.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 150 units of AscI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 0% 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
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,192,676
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