Difference between revisions of "Production of LentiCRISPR Viruses"
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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: | ||
{| class="wikitable" | {| class="wikitable" | ||
| Line 47: | Line 50: | ||
* 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. | ||
Revision as of 16:19, 8 December 2017
This protocol is modified from the Addgene website protocol
Materials
- Use the 293T/17 cells (purchased as a stock from ATCC and aliquoted in separate box in the liquid nitrogen; ATCC page).
- Plasmids (lenti-vector, psPAX2, pMD2.G)
- OptiMEM
- Chloroquine stocks (25 mM stocks). See Preparation of Chloroquine Stocks.
- PEI (1 mg/mL stocks). See Preparation of PEI Stocks.
- DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG
Preparation of Cells
- Thaw and passage cells. Use passage <20. These amounts are for one plasmid.
- Split cells normally each time seeding 7 x 105 cells in a 10 cm dish, in DMEM/PSG/10% FBS.
- The day before the transfection, seed 3.8 x 106 cells into a fresh plate. Seed cells into media without PSG.
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:
| Reagent | pmoles | ug | Volume Added |
|---|---|---|---|
| psPAX2 | 1.3 | 9.2 | |
| pMD2.G | 0.72 | 2.77 | |
| LentiCRISPRv2-sgRNA | 1.64 | 16.1 | |
| OptiMEM | - | - | 500 uL |
- For this amount of DNA (28 ug) try using 3:1 PEI:DNA, so add 84 uL PEI into 1 mL OptiMEM.
- Gently add the PEI solution dropwise into the DNA solution.
- Incubate at room temperature for 15-20min.
- Add transfection mixture slowly to the cells.
- 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.
- Centrifuge at 500g for 5 minutes to pellet any cells.
- Filter supernatant through the 0.45 um. Aliquot and snap freeze in liquid nitrogen in screw capped vials.
