Nb.BbvCI is a nicking endonuclease that cleaves only one strand of a dsDNA substrate. It introduces a nick on dsDNA substrates without cleaving the double helix, commonly used in isothermal amplification techniques such as SDA and RCA. The nick generated by Nb.BbvCI creates a DNA gap, initiating the strand-displacement activity of DNA polymerase, and enabling repeated cycles of nicking, displacement, and extension, thereby achieving exponential amplification of nucleic acids. Specifications Enzyme Nb.BbvCI Cat.NO. 14232ES75 / 14232ES92 Size 300 U / 10000 U Recognition Site 5'-CCTCA GC-3' 3'-GGAGT↑CG-5' Enzyme Activity 10 U/μL Recommended Reaction Conditions 1× Nb.BbvCI Buffer; incubate at 37°C. Inactivation Conditions Incubate at 80°C for 20 minutes. Components Components No. Name 14232ES75 14232ES92 14232-A Nb.BbvCI(10 U/μL) 30 μL 1 mL 14232-B 10× FuniCut™ Buffer 1 mL 6 mL 14232-C 10× FuniCut™ Color Buffer 1 mL 6 mL Unit Definition One unit (U) is defined as the amount of enzyme required to completely convert 1 µg of supercoiled p615 DNA into the open circular form in 50 µl of reaction mixture within 1 hour at 37°C. Notes 1. Do not exceed 10% of the total reaction volume when adding enzyme to avoid star activity caused by excessive glycerol. 2. Additives in restriction enzyme storage buffers (e.g., salts, EDTA, or ethanol) may inhibit the reaction. The smaller the reaction volume, the stronger the inhibitory effect, especially when contaminants (e.g., glycerol, salts) from the enzyme storage buffer mix with the substrate solution. 3. For your safety and health, wear a lab coat and disposable gloves. 4. This product is intended for research use only. Instructions 1. DNA Digestion Protocol 1.1 Prepare the reaction mixture on ice using the following recommended order: Reagent Volume ddH₂O to 50 µl 10× CutOne™ Buffer or 10× CutOne™ Color Buffer 5 µl Substrate DNAa 1 µg Nb.BbvCI (10 U/µl) 1 µl Total 50 µl [Note]:a. Phenol, chloroform, ethanol, EDTA, detergents, or high salt concentrations in the DNA substrate may inhibit Nb.BbvCI activity. 1.2 Mix gently by pipetting or tapping the tube (do not vortex), then briefly centrifuge to collect droplets on the tube wall. 1.3 Incubate at 37°C for 15 min to 3 h. 1.4 Stop the reaction by incubating at 80°C for 20 min to inactivate the enzyme, or alternatively, terminate the reaction by column purification or phenol/chloroform extraction. 2.Number of Nb.BbvCI Recognition Sites in Different DNA Substrates DNA Substrate λDNA ΦX174 pBR322 pUC57 pUC18/19 SV40 M13mp18/19 Adeno2 Number of Sites 7 3 0 0 0 0 2 9 3. Effect of Methylation Modifications Methylation Type Dam Dcm EcoKI EcoBI Effect on Nb.BbvCI Activity No effect No effect No effect Cleavage blocked
