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Restriction Endonucleases >
Restriction Endonucleases >
BpuEI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
Bacillus pumilus (C. Nkenfou)
Reagents Supplied:
NEBuffer 2
S-adenosylmethionine (SAM)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 10% | | NEBuffer 4: | | 75% |
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 |
Activity at 37°C:
20%
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 2
Supplemented with 80 μM S-adenosylmethionine (SAM)
Incubate at
25°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 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 25°C in a total reaction volume of 50 µl.
Concentration:
1,000 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 7.5 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- Overdigestions with > 4 units of BpuEI per µg of DNA and incubations > 1 hour are not recommended.
- Storage: For long term storage (>6 months), store at -80°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 BpuEI, > 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 BpuEI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 10 units of BpuEI incubated for 16 hours at 25ºC
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 BpuEI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 25ºC
released < 0.3% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 3 units of BpuEI with 1 μg of
mp18 RF I DNA for 4 hours at 25ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Reagents Sold Separately
NEBuffer 2
S-adenosylmethionine (SAM)
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