 |
|
 |
|
|
| Home >
Products >
Restriction Endonucleases >
Restriction Endonucleases >
AfeI |
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned AfeI gene from Alcaligenes faecalis T2774 (S.K. Degtyarev)
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 50% | | 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: Blocked | |
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 pXba DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
pXba 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:- AfeI is an isoschizomer of Eco47III.
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 AfeI, > 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 AfeI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of pXba
and 20 units of AfeI 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 AfeI 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.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of AfeI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 20% conversion to RFII as determined by agarose gel electrophoresis.
Reagents Sold Separately
NEBuffer 4
|
|
|
 |
|
New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
|
 |
|
|
