The AapCas12b nuclease (also known as C2c1) is a DNA endonuclease mediated by tracrRNA:crRNA (or sgRNA), derived from the thermophilic bacterium Alicyclobacillus acidophilus. In the presence of a PAM (TTN) sequence at the target double-stranded DNA (dsDNA), it specifically cleaves the target dsDNA, causing double-strand breaks and generating sticky ends. AapCas12b can specifically cleave single-stranded DNA targets independent of the PAM sequence. Both double-stranded and single-stranded DNA targets can activate the trans-cleavage activity of AapCas12b. When the AapCas12b enzyme forms a ternary complex with sgRNA and target DNA, it becomes activated for nonspecific ssDNA trans-cleavage activity, cutting any arbitrary sequence of ssDNA in the system. The optimal cleavage reaction temperature for AapCas12b is 60oC, which makes it more heat-resistant than AacCas12b, and more suitable for use with LAMP to develop isothermal amplification/CRISPR-Cas detection systems. Features Broad reaction temperature range: Exhibits cleavage activity within the temperature range of 37~65oC Low nuclease residue: No residual exonuclease, nickase, or RNase Cis-cleavage activity: Highly Effective Cleavage of Double-Stranded DNA in Vitro Trans-cleavage activity: High trans-cleavage activity, suitable for nucleic acid detection Applications CRISPR/Cas gene editing Diagnostic and detection based on the CRISPR/Cas system Other detection applications combined with isothermal nucleic acid amplification technologies (RPA and LAMP), etc Specifications Molecular weight 130 KDa Concentration 10 µM Purity >95% (SDS-PAGE) Unit Definition The quantity of Cas12b enzyme required to cleave 1 pmol of ssDNA probe within 1 minute at 60oC in a total reaction volume of 20 μL, containing 1×reaction buffer, is defined as 1 U Components Components No. Name 14808ES65 14808ES80 14808-A AapCas12b Nuclease (10 μM) 10 μL 100 μL 14808-B 10×Reaction buffer 1 mL 1 mL Shipping and Storage This product should be stored at -25 ~ -15oC for 2 years. Figures Figure 1. Cis-cleavage Activity Verification of AapCas12b Nuclease Note: In a 20 μL reaction system containing Target dsDNA with a PAM sequence, sgRNA, Cas12b, and 1×reaction buffer, the mixture was incubated at 60oC for 30 minutes and then inactivated at 85oC for 5 minutes. The agarose gel electrophoresis results showed that three batches of AapCas12b Nuclease were all capable of effectively cleaving the dsDNA, with cleavage efficiency comparable to that of the imported brand T*. Figure 2. AapCas12b Nuclease Trans-cleavage Activity Test Results Note: In a 20 μL reaction system containing Target dsDNA with a PAM sequence, sgRNA, Cas12b, Report ssDNA fluorescent probe, and 1×reaction buffer, the mixture was incubated at 60oC for 1 hour, and the fluorescence signal was collected every 30 seconds. The results showed that in the presence of Target DNA, sgRNA, and Cas12b together, the Report ssDNA fluorescent probe could be cleaved, thereby releasing the fluorescence signal. Documents: Safety Data Sheet 14808_MSDS_Ver.EN20241209.pdf Manuals 14808_Manual_Ver.EN20241209.pdf
