BspEI is a Type IIP restriction enzyme originally isolated from Bacillus spp. and produced via recombinant expression and purification in Escherichia coli. BspEI recognizes and cleaves the sequence TCCGGA, generating a 5′-overhanging cohesive end with a 4-nucleotide protrusion. BspEI requires its dedicated buffer, Cut Buffer C, and is not compatible with the universal buffer FuniCutTM Buffer. Specifications Cat.No. 15230ES76 Unit Size 500 U Recognition Site 5'-T↓CCGGA-3' 3'-AGGCC↑T-5' Reaction Conditions 1×Cut Buffer C; incubate at 37°C Inactivation Incubate at 80°C for 20 min Unit Definition One unit (U) is defined as the amount of enzyme required to completely digest 1 μg of λ DNA in 50 μL reaction volume in 1 hour at 37°C. Isoschizomer Kpn2I, MroI, AccIII, Aor13HI, Bsp13I, BseAI Components Components No. Name 15230ES76 15230-A BspEI (10 U/μL) 50 μL 15230-B 10× Cut Buffer C 1 mL Storage This product should be stored at -25~-15℃ for 2 years. Instructions 1. DNA Digestion Protocol 1)Prepare the reaction mixture on ice following the recommended component addition order below: Components Volume ddH2O To 50 μL 10× Cut Buffer C 5 μL Substrate DNA* 1 μg BspEI (10 U/μL) 1 μL Total 50 μL [Note]: The DNA substrate must be free of phenol, chloroform, ethanol, EDTA, detergents, or high salt concentrations, as these may inhibit BspEI activity. 2)Gently mix by pipetting up and down or flicking the tube wall (do not vortex), then briefly centrifuge to collect droplets from the tube walls. 3)Incubate at 37°C for 15 min ~ 3 h. 4)Inactivate the enzyme by incubating at 80°C for 20 min to stop the reaction, or terminate by purification using a spin column or phenol/chloroform extraction. 2. Number of Recognition Sites in Different Genomes λDNA ΦX174 pBR322 pUC57 pUC18/19 SV40 M13mp18/19 Adeno2 24 0 1 0 0 0 0 8 3. Effect of Methylation Dam Dcm CpG EcoKI EcoBI Cleavage blocked No effect Cleavage affected No impact No impact 4. Activity in Different Reaction Buffers* Reaction buffer FuniCutTM Buffer(Yeasen) Thermo Scientific FastDigest Buffer NEB CutSmart® Buffer Takara QuickCut™ Buffer Activity
