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Restriction Endonucleases >
Restriction Endonucleases >
ApeKI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
E. coli strain that carries the ApeKI gene from Aeropyrum pernix K1 (ATCC 700893).
Reagents Supplied:
NEBuffer 3
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 75% | | NEBuffer 3: | | 100% | | NEBuffer 4: | | 50% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Activity at 37°C:
10%
Heat Inactivation:
No
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 3
Incubate at
75°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
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 75°C in a total reaction volume of 50 μl.
Concentration:
4,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.9 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- ApeKI is an isoschizomer of TseI.
- ApeKI is a highly thermostable restriction enzyme that can survive temperatures as high as 95°C. At that temperature, the half-life of the enzyme is 20 minutes.
Usage notes:- Exonuclease activity quality control was performed at 37°C to detect any E. coli contaminants which are not active at 75°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 ApeKI, > 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 ApeKI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA
and 9 units of ApeKI incubated for 16 hours at 75ºC
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 20 units of ApeKI 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 3
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