Introduction

SpCas9 is a DNA endonuclease guided by crRNA and tracrRNA (or sgRNA alone), which is derived from S. pyogenes. SpCas9 specifically recognizes and cleaves dsDNA target in a PAM (NGG)-dependent manner. The cleavage site of Cas9 in the target sequence is 3 bps away from the PAM site. In addition, SpCas9 can also specifically cleave ssDNA or ssRNA in the presence of DNA PAMmer sequences (for details, please refer to PMID: 24476820; PMID: 25274302). SpCas9 can also be used in in vitro experiments for cleaving target DNA and cloning target fragments (for details, please refer to PMID: 26323354).

This product is lyophilized glycerol-free SpCas9 nuclease, which can be shipped and stored at room temperature. After being dissolved with the Reconstitution Solution, it shows the same performance as the non-lyophilized type of SpCas12b Nuclease (#32101, 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

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

Components

· It is recommended to use 124 μL of reconstitution solution to reconstitute 100 μg SpCas9 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

SpCas9 (Cat# V32101E) CRISPR Cis-cleavage Experiment

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

2.Put the reaction mixture at 37℃for 30 min and then inactive the enzyme at 85℃for 5 min . Analyze products using nucleic acid electrophoresis.

Experimental Results