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Restriction Endonucleases >
Restriction Endonucleases >
EcoP15I |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned EcoP15 l res-mod genes in plasmid pSHl180 (DN. Rao)
Reagents Supplied:
NEBuffer 3 (10X)
ATP (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 100% | | 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 |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 1.00 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Supplemented with 100 μg/ml Bovine Serum Albumin and 1 mM ATP
Incubate at
37°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 enzyme required to digest 1 µg of pUC19 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
pUC19 DNA
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
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:- ATP is required for DNA cleavage. Efficient cleavage requires the presence of two inversely oriented recognition sites. A head to head orientation is preferred. The "head" of the sequence is defined as the dG at the 3´ end of the CAGCAG strand (top strand). Cleavage efficiency is also affected by the distance between the two sites.
- Not sensitive to dam, dcm or mammalian CpG methylatiopn.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of EcoP15I 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 50 units of EcoP15I 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 25 units of EcoP15I with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Reagents Sold Separately
NEBuffer 3
BSA
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