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Restriction Endonucleases >
Restriction Endonucleases >
BsrFI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the BsrFI gene from Bacillus stearothermophilus CPW16 (Z. Chen).
Reagents Supplied:
NEBuffer BsrFI
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 75% | | 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: Blocked |
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 BsrFI
Incubate at
37°C.
1X NEBuffer BsrFI:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025 % Triton X-100
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
pBR322 DNA
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
- BsrFI is an isoschizomer of Cfr10I.
- The recommended reaction buffer for BsrFI has changed from NEBuffer 2 + BSA to NEBuffer BsrFI (without BSA). Either reaction buffer produces 100% activity. NEBuffer BsrFI is preferred because it minimizes star activity. The composition of NEBuffer BsrFI is identical to NEBuffer EcoRI and NEBuffer SspI.
- BsrFI can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis. To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.
- Fragments produced by noncanonical cleavage due to star activity may be observed with 1 unit of enzyme in similar conditions.
FAQs
- Do degenerate recognition sites need to be palindromic?
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 BsrFI, > 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 BsrFI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 10 units of BsrFI 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 10 units of BsrFI 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 15 units of BsrFI 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 BsrFI
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 6,066,487
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