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Restriction Endonucleases >
Restriction Endonucleases >
AlwI |
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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 AlwI gene from Acinetobacter lwoffii (R. Morgan).
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 100% | | 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: Blocked | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+) Intermediate activity. Suitable for extended digestion, but < 8 hours.
More information about: Extended Digests with Restriction Enzymes
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 (dam-) in 1 hour at 37°C in total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
λ DNA (dam-)
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:- AlwI produces DNA fragments that have a single-base 5´ extension which are more difficult to ligate than blunt-ended fragments.
FAQs
- Is AlwI blocked by dam methylation?
- Why is an extended digest not recommended?
- Why is AlwI- cut DNA difficult to ligate?
- How can I increase ligation efficiency?
- What is the activity of AlwI 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 2-fold overdigestion with AlwI, 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 AlwI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 25 units of AlwI 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 25 units of AlwI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
Companion Products
dam-/dcm- Competent E. coli
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