Production of LentiCRISPR Viruses: Difference between revisions
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* Thaw and passage cells. Use passage <20. These amounts are for one plasmid. | * Thaw and passage cells. Use passage <20. These amounts are for one plasmid. | ||
* Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS. | * Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS. | ||
* The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate. Seed cells into media '''without PSG''' | * The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate. Seed cells into media '''without PSG'''. | ||
* Prepare DNA according to this table in a sterile 2 mL tube | |||
==Transfection Procedure== | |||
* The morning of the transfection day, replace the media with fresh DMEM '''without PSG''' and containing 10 uL of 25 mM chloroquine. Wait ~5h before going onto the next step. | |||
* Prepare DNA according to this table in a sterile 2 mL tube: | |||
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* Add transfection mixture slowly to the cells. | * Add transfection mixture slowly to the cells. | ||
* Incubate overnight. The next day carefully replace with media containing PSG. | * Incubate overnight. The next day carefully replace with media containing PSG. | ||
==Collecting Viral Particles== | |||
* Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube. | * Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube. | ||
* Centrifuge at 500g for 5 minutes to pellet any cells. | * Centrifuge at 500g for 5 minutes to pellet any cells. | ||