Description

AapCas12b (previously known as C2c1) is a DNA endonuclease mediated by tracrRNA and crRNA (or sgRNA), which specifically cleaves the dsDNA target in a PAM (TTN)-dependent manner, generating DSBs with sticky ends, and cleave ssDNA target in a PAM-independent manner. In addition, both dsDNA and ssDNA targets can trigger the trans-cleavage activity of AapCas12b, that is, when the AapCas12b binds sgRNA and target DNA to form a ternary complex, the trans-cleavage activity of AapCas12b will be released to non-specifically cleave ssDNA sequences in the reaction system. AapCas12b is derived from Alicyclobacillus acidophilus and produced from E. coli BL21(DE3), with the optimal reaction temperature of 60 °C for in vitro analysis. Therefore, AapCas12b is more suitable for the creature of a “One-Step” CRISPR-Cas detection system when combined with the LAMP isothermal amplification technology.

This product is lyophilized glycerol-free AapCas12b nuclease, which can be shipped and stored at room temperature. After being dissolved with its reconstitution buffer, it shows the same performance as the non-lyophilized type of AapCas12b (C2c1) Nuclease (#32118, ToloBio). This product can be provided in a variety of lyophilized forms such as lyophilized powder and lyophilized beads to meet the needs of different downstream applications

Storage Conditions

This product is stored and shipped at room temperature. After reconstitution, the liquid enzyme should be stored at -20 °C or below and is stable for at least one month. Repeated freezing and thawing should be avoided for reconstituted liquid enzyme

Components

· 100 μg of AapCas12b nuclease = 750 pmol. It is recommended to use 150 μL of reconstitution solution to reconstitute 100 μg AapCas12b lyophilized powder, generating 5 μM (5 pmol/μL) liquid enzyme. Once dissolved, the liquid enzyme should be stored at -20 °C or below.

Experiment Procedure

AapCas12b (Cat # V32118E) CRISPR Trans-cleavage Experiment

1.Prepare the reaction mixture of CRISPR Trans-cleavage Experiment on ice as below.

2.Put the reaction mixture in the real-time PCR instrument to detect the fluorescent signal at 60 °C, and collectsthe fluorescent signals every 30 sec.

Experimental Results