FAQs for BstUI Enzyme Finder Double Digest NEBcutter REBASE

BstUI Catalog # Size Concentration R0518L 5,000 units 10,000 units/ml R0518S 1,000 units 10,000 units/ml Download: MSDS PDF Recognition Site:  | single letter code Source: Bacillus stearothermophilus U458 (Z. Chen) Reagents Supplied: NEBuffer 4 (10X) Enzyme Properties Activity in NEBuffers: NEBuffer 1: 100% NEBuffer 2: 100% NEBuffer 3: 50% 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: Blocked More information about: Methylation Sensitivity Activity at 37°C: 20% Heat Inactivation: No Survival in a Reaction: (+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours. More information about: Extended Digests with Restriction Enzymes Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Incubate at 60°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 60°C in 50 µl assay buffer. Concentration: 10,000 units/ml Unit Assay Substrate: λ DNA Storage Conditions: 10 mM Tris-HCl 50 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: BstUI is an isoschizomer of FnuDII. FAQs The NEB catalog has historically stated that activity in NEBuffer 4 is less than 100% yet this enzyme is now supplied with NEBuffer 4. What have you changed? Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme? If I have an old tube of enzyme, what NEBuffer should I use? Will the new enzyme work in the originally supplied NEBuffer? Why is NEB switching this restriction enzyme to NEBuffer 4? How can this enzyme be inactivated? 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 20-fold overdigestion with BstUI, approximately 50% 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 BstUI. 16-Hour Incubation: A 50 μl reaction containing 1 μg of DNA and 300 units of BstUI incubated for 16 hours at 60ºC 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 200 units of BstUI with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 60ºC released < 0.1% of the total radioactivity. Reagents Sold Separately NEBuffer 4