Description
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 60°C, 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°C～65°C.
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 60°C in a total reaction volume of 20 μL, containing 1 × reaction buffer, is defined as 1 U. |
*: The concentration of AapCas12b Nuclease is 10 μM, which is equivalent to 10 pmol/μL.
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~-15℃ 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 60°C for 30 minutes and then inactivated at 85°C 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 60°C 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.
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