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Restriction Endonucleases >
Restriction Endonucleases >
BfaI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
Bacteroides fragilis (VPI 3392)
Reagents Supplied:
NEBuffer 4
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 50% | | 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: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
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 in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
For long term storage (>30 days), store at -70°C.
Diluent Compatibility:
Diluent B
Notes
General notes:- BfaI is an isoschizomer of Mae I and Rma I.
- BfaI has shown minimal cleavage of unpurified PCR products. Therefore, PCR products should be purified prior to BfaI digestion in 1X NEBuffer 4.
FAQs
- What are the recommended storage conditions for BfaI?
- Does BfaI work well on PCR products?
- Does BfaI replace an enzyme previously sold?
- What is the activity of BfaI at 25°C?
- Are overnight digestions with BfaI recommended?
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 BfaI, < 5%
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 BfaI.
Note: This ligation efficiency was not increased by using a lower overdigestion. The relatively low efficiency is due to the character of the overhang left by Bfa I, not due to any contaminant.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 200 units of BfaI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Note that the high enzyme concentration described in this assay may result in star activity.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 500 units of BfaI 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
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