Bbs I is a Type IIS restriction endonuclease encoded by the BbsI gene of Brevibacillus laterosporus. FuniCut Series Endonucleases are fast restriction endonucleases that can accurately complete DNA cleavage within 5 to 15 minutes using genetic engineering recombination technology. They are suitable for rapid cleavage of plasmid DNA, PCR products, genomic DNA, etc. FuniCut rapid endonucleases share a common enzyme cleavage buffer, simplifying the enzyme reaction system. In addition, they have good enzyme activity redundancy and can easily handle the enzymatic cleavage of excess substrates or difficult templates. Specifications Cat.No. 15070ES25 Size 25T Restriction Enzyme Cut Site 5'-GAAGAC(N)₂↓-3'3'-CTTCTG(N)₆↑-5' Recommend reaction conditions 1×FuniCut™ Buffer, Incubate at 37°C Enzyme activity 20 U/μL Inactivation condition Incubate at 80℃ for 20 min Isoschizomer BstV2I, BpiI Components Components No. Name 15070ES25 (25T) 15070-A FuniCut™ Bbs I 25 μL 15070-B 10×FuniCut™ Buffer 1 mL 15070-C 10×FuniCut™ Color Buffer* 1 mL 【Note】: *10×FuniCut Color Buffer contains red and yellow tracking dyes which allow the product to be used directly for gel electrophoresis. The red dye in FuniCut Color Buffer has a migration rate similar to a 2500 bp double-stranded DNA fragment in 1% agarose gel; the yellow dye has a migration rate similar to a 10 bp double-stranded DNA fragment in 1% agarose gel. Storage This product should be stored at -25~-15℃ for 2 years. Notes 1. Star activity was not observed after 3 h incubation, but delayed enzyme digestion may result in star activity. 2. This product is for research use only. 3. Please operate with lab coats and disposable gloves，for your safety. Instructions 1. DNA rapid enzyme digestion protocol 1) Prepare the system reaction solution using the following recommended sample addition sequence (operate on ice) Components Plasmid DNA PCR products gDNA ddH2O 15 μL 16 μL 30 μL 10×FuniCut Buffer or 10×FuniCut Color Buffer 2 μL 3 μL* 5 μL Substrate DNA 2 μL (~1 μg) 10 μL (~0.2 μg) 10 μL (5 μg) FuniCut Bbs I 1 μL 1 μL 5 μL Total 20 μL 30 μL 50 μL 【Note】：*This system refers to purified PCR products. Unpurified PCR products have a certain ionic strength and the amount of 10×FuniCut Buffer added can be reduced accordingly to 2 μL. If cloning and other experiments are performed in the next step, the PCR product must be purified before enzyme digestion. 2) Mix by gentle pipetting or swirling (do not vortex), then centrifuge briefly to collect the droplets. 3) Incubate at 37°C for 15 min (plasmid), 15-30 min (PCR product) or 30-60 min (genomic DNA). 4) Incubate at 80°C for 20 min to inactivate the enzyme and stop the reaction (optional). 5) If FuniCut Colour Buffer is used for the digestion reaction, the product can be loaded directly onto the electrophoresis. 2. Double or multiple enzyme digestion protocol 1) The dosage of each endonuclease is 1 µL and the reaction system should be expanded accordingly as needed. 2) The total volume of all endonucleases should not exceed 1/10 of the total reaction system. 3) If the optimal reaction temperatures of the selected endonucleases are different, use the enzyme with the lowest optimal temperature for enzyme digestion, then add the enzyme with the highest optimal temperature and incubate at a higher temperature. 3. Reaction system Components Volume（20 μL） Volume（20 μL） Volume（50 μL）* DNA 1 μg 2 μg 5 μg 10×FuniCut Buffer or 10×FuniCut Color Buffer 2 μL 2 μL 5 μL FuniCut Bbs I 1 μL 2 μL 5 μL Total 20 μL 20 μL 50 μL 【Note】:*If the total reaction volume is greater than 20 μL, use a water, metal or sand bath and increase the incubation time. 4. Number of recognition sites in different DNA λDNA ΦX174 pBR322 pUC57 pUC18/19 SV40 M13mp18/19 Adeno2 24 3 3 0 0 3 0 27 5. Effects of methylation modification Dam Dcm CpG EcoKI EcoBI No influence No influence No influence No influence No influence 6. Activity in different reaction buffers* Reaction buffer FuniCut™ Buffer Thermo FastDigest Buffer NEB CutSmart™ Buffer Takara QuickCut™ Buffer Activity 100% ≤10% 100% 25%
