AapCas12b is a Cas nuclease of the Type V subtype, with robust activity between 37°C and 60°C. Guided by crRNA and tracrRNA (or sgRNA formed by the two), it exhibits cis-cleavage activity against both double-stranded and single-stranded DNA targets. For double-stranded DNA targets, AapCas12b recognizes the position complementary to the spacer sequence of crRNA (or sgRNA) downstream of the PAM sequence (5'-TTN) in the target DNA, and specifically cleaves the target double-stranded DNA to generate sticky ends. For single-stranded DNA targets, its specific cleavage is independent of the PAM sequence. After forming a ternary complex with the target DNA and sgRNA, AapCas12b also exhibits trans-cleavage activity, i.e., non-specific cleavage of single-stranded DNA of any sequence. AapCas12b also shows good activity in common isothermal amplification reaction buffers.
Components List
| A1522667 | Components | Appearance | 100pmol | 1000pmol | Storage | | A1522667A | AapCas12b (10 μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | A1522667B | 10× Cas12b Buffer | Liquid | 1ml | 1ml | -20℃. Avoid freeze/thaw cycle. |
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Quality Control
- Protein PurityDetermined by SDS-PAGE, the protein purity is ≥95%.
- Endonuclease Residue Detection10 pmol of AapCas12b is incubated with 200 ng of supercoiled plasmid DNA at 37℃ for 4 h. As determined by agarose gel electrophoresis, less than 10% of the plasmid DNA is converted to nicked or linear form.
- Non-specific Nuclease Residue Detection10 pmol of AapCas12b is incubated with 15 ng of double-stranded DNA fragments at 37℃ for 16 h. No change in the double-stranded DNA substrate is observed by agarose gel electrophoresis.
- RNase Residue Detection10 pmol of AapCas12b is incubated with 500 ng of total RNA at 37℃ for 1 h. More than 90% of the RNA remains intact as determined by agarose gel electrophoresis.
- Host DNA Residue DetectionUsing E. coli 16S rDNA-specific primer-probe set, the residual host genomic DNA in 10 pmol of AapCas12b is detected by qPCR and is less than 1 copy.
Inactivation Condition
Incubate at 85℃ for 5 min.
Protocol
1. Cis Cleavage Assay
① Prepare the following reaction system on ice:
| Reagent | Volume | Final Concentration |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.5 μl | 250 nM | | sgRNA (10 μM)ᵃ | 0.5 μl | 250 nM | | Target DNA (1 μM)ᵃ | 0.5 μl | 25 nM | | Nuclease-free water | To 20 μl | — |
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ᵃ. If the cleavage products are detected by gel electrophoresis, it is recommended to use double-stranded DNA as the target, with a length between 300 bp and 3 kb. It is recommended that the molar ratio of AapCas12b : sgRNA : Target DNA is 10:10:1 to ensure complete cleavage of the target DNA as much as possible.
② Incubate at 60℃ for 30 min~1 h, then inactivate at 85℃ for 5 min.
③ Analyze the products by agarose gel electrophoresis. To avoid abnormal electrophoresis caused by AapCas12b binding to DNA, ensure heat inactivation is performed. If electrophoresis abnormalities still occur, treat the products with Proteinase K before electrophoresis analysis.
2. Trans Cleavage Assay
① Prepare the following reaction system on ice:
| Reagent | Volume | Final Concentration |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | sgRNA (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Target DNA (1 μM)ᵇ | 0.5~5 μl | 25~250 nM | | ssDNA Probe (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Nuclease-free water | To 20 μl | — |
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ᵃ. The amount of each component in the trans-cleavage assay can be adjusted according to the experimental purpose. When using small amounts, it is recommended to prepare a master mix by dilution first.
ᵇ. The target DNA in the trans-cleavage assay can be single-stranded DNA or double-stranded DNA with a PAM sequence.
② Place the reaction in a qPCR instrument, select the corresponding channel according to the fluorophore of the ssDNA probe, and perform a constant temperature reaction at 60℃, with each cycle set to 30 s. The number of cycles can be set to 30~60.
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