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RNP assembly for HDR reaction
Last updated date: Apr 13, 2020 Views: 1192 Forks: 0
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RNP assembly for HDR reaction with RNAiMAX
- Make a 9 µM working solution of sgRNA by diluting the stock with nuclease-free water.
- Make a 3 µM working solution of Cas9 /Cas9 Mutants by diluting with PBS (sterile) or Opti-MEM.
- Make a 3 µM working solution of DBCO-adaptor by diluting the stock with nuclease-free water.
- Make a 3 µM working solution of ssODN by diluting the stock with nuclease-free water.
- Form the RNP complexes as follows below:
Component | Single reaction | x3.3 (triplicates) |
Cas9(3 μM ) | 0.5μl | 1.65 μl |
sgRNA(9 μM ) | 0.5μl | 1.65 μl |
Opti-MEM | 10.3μl | 33.4μl |
Incubate at room temperature for 5min | ||
DBCO-adaptor(3 µM ) | 0.6μl | 1.98μl |
Incubate at 4 degree for 3 hours with shaking | ||
ssODN | 0.6μl | 1.98μl |
Total | 12.5μl | 41.25μl |
6. Form the liposome complexes as follows below. You can make a master mix of the RNAiMAX and Optimem and add this directly to the RNP tube from above.
Component | Single reaction | x3.3 (triplicates) |
RNP | 12.5μl | 41.25μl |
RNAiMAX | 1.2 μl | 3.96 μl |
Opti-MEM | 11.3μl | 37.3μl |
Total | 25μl | 82.5μl |
7. Gently mix the reaction and incubate at room temperature for 20 minutes.
8. Added the mixture into the medium (for 96-well transfection).
- Ling, X and Liu, T(2020). RNP assembly for HDR reaction. Bio-protocol Preprint. bio-protocol.org/prep271.
- Ling, X., Xie, B., Gao, X., Chang, L., Zheng, W., Chen, H., Huang, Y., Tan, L., Li, M. and Liu, T.(2020). Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates . Science Advances 6(15). DOI: 10.1126/sciadv.aaz0051
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