For molar concentration and other related product information, please refer to: CRISPR/Cas9 related enzyme proteins, antibodies, and other reagents
RNP Complex Formation and Target DNA binding
1. Add the following components to a sterile, nuclease-free tube on ice:
Components | Volume | Final Concentration | |
sgRNA (300 nM) | 2 µl | ~30 nM | |
dCas9 Nuclease Protein (1000 nM) | 0.60 µl | ~30 nM | |
10X Cas9 Nuclease Reaction Buffer | 2 μl | 1X | |
Nuclease-free H2O | 12.4 μl | - | |
Pre-Incubate for 15 minutes at room temperature | |||
Substrate DNA (30 nM) | 3 μl | 3 nM | |
Total Volume | 20 μl | - | |
2. Collect all components by a brief centrifugation. Incubate the reaction at 37 °C for 1 hour.
References:
1. Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA Amin Aalipour et al. Clinical Chemistry 2018 Vol. 64, Issue 2 p307-p9162. Purified Cas9 Fusion Proteins for Advanced Genome Manipulation Jovan Mircetic et al. Small Methods 2017 1, 16000523. Disruptive non-disruptive applications of CRISPR/Cas9 Jonathan LSchmid-Burgk Current Opinion in Biotechnology December 2017, Volume 48 Pages 203-2094. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins Toshitsugu Fujita et al. Genes to Cells Volume 21, Issue 4 April 2016 Pages 370–3775. High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Evan A Boyle et al. PNAS 2017 May, 114 (21) 5461-54666.CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells Wulan Deng et al. PNAS 2015 vol. 112 | no. 38 p11870-11875
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