This product is free from RNase, DNA exonuclease, and non-gRNA-dependent DNA endonuclease.concentration: 1μM(158ng/μl).
Stability And Storage
Stored at -80ºC for long-term storage.
Storage Temp
Store at -20°C
Shipped In
Ice chest + Ice pads
Product Description
Aladdin's SpCas9 is a Streptococcus Pyogenes Cas9 Nuclease, recombinantly expressed in E.coli and purified by the PerfectProtein™ technology platform. It is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein.CRISPR/Cas9-based genome editing technology is a new breakthrough, which has been widely used in genome engineering. CRISPR (clustered regularly interspaced short palindromic Repeats) is an adaptive immune system for gene silencing of exogenous phage or viral nucleic acid by using RNA-guided DNA nuclease Cas9, on which the CRISPR/Cas9-based gene-editing technique has been developed to be widely used in prokaryotes and eukaryotes. This technology enables site-specific cleavage of targeted genomic DNA by gRNA-guided Cas9 and introduction of frameshift mutation at the cutting site by error-prone repair or homologous recombination, during which gRNA ensures the specificity of the cleavage site. With the development of CRISPR technology, the technology can not only achieve gene knockout, but also achieve a variety of mutations such as point mutation and insertion mutation, especially in the clinical application can be used to repair undesirable mutations, etc [1, 2]. Moreover, transcriptional activation or inhibition of target genes can be achieved by recruiting transcriptional activation or transcriptional directly or indirectly via the Cas9 mutant without endonuclease activity (dCas9).CRISPR/Cas9 system is composed of Cas9 Nuclease and gRNA. The gRNA consists of two parts
Source：
Recombinant SpCas9 expressed in E. coli.
Precautions：
We strongly recommend wearing a disposable mask and using nuclease-free tubes and reagents to avoid RNase contamination.We recommend using DNase and DNase Away to remove nucleases on the surface of benches, pipettes, or other equipment. RNase Inhibitor can also be used to protect RNA from degradation.This kit is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1. Thaw all reagents at room temperature and keep on ice.2. Prepare 300nM gRNA and 30nM substrate DNA by diluting the stock with nuclease-free water on ice.3. Assemble the reaction in the following order:ReagentVolumeNuclease-free Water20µlCas9 Reaction Buffer(10X)3µlgRNA (300nM)3µlCas9 Nuclease(1μM)1µlReaction Volume27µl4. Mix by gently pipetting or vortexing and pulse-spin in a microfuge. Incubate at 25ºC for 10 minutes.5. Add 3µl of 30nM substrate DNA, mix gently, and pulse-spin in a microfuge. 6. Incubate at 35ºC for 15 minutes. If necessary, prolong the incubation time to 30-120 minutes.7. Add 3µl of Proteinase K . Mix gently and incubate at 25ºC for 10 minutes.8. Proceed with fragment analysis by adding 6µl of 6X DNA loading buffer followed by agarose gel electrophoresis. The reaction can also be stored at -20ºC for future analysis.FAQ:1. The substrate DNA is digested incompletely.a. Incomplete digestion may be caused by an incorrect ratio of Cas9 Nuclease, sgRNA, and target site. For complete digestion, we recommend a 10:10:1 molar ratio of Cas9 Nuclease:sgRNA:target site. b. Incomplete digestion may be caused by inappropriate sgRNA sequences. Redesign the sgRNA sequence.c. Incomplete digestion may be caused by the degradation of sgRNA. Check the integrity of the sgRNA by gel electrophoresis.References：1. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Cell. 2014.156(5):935-49.2. Marangi M, Pistritto G. Front Pharmacol. 2018. 9:396.
