Plate approximately 250,000 S293T cells per well in a 6-well plate. Alternatively, you can plate about 300,000 cells early on Tuesday morning and transfect late in the afternoon. Sometimes I forget J
- Warm FuGene reagent to room temp (10-15 minutes prior to use).
- Add calculated amount of serum-free media to sterile Eppendorf tube (for a 6-well plate, should contain 100ul total including DNA and FuGene Reagent).
- Add calculated amount of DNA.
600ng target DNA (shRNA construct)
400ng delta VPR8.2
- Add FuGene reagent directly to media. For a 6-well plate in 3 ml of serum-free media, use 3ul FuGene/~1ug DNA. Swirl with pipet tip. DO NOT PIPET UP AND DOWN AND AVOID ADDING FUGENE TO SIDES OF TUBE.
- Allow to incubate at room temp for 15 minutes.
- Add dropwise to plate.
- you may also want to plate your target cells this day. See Wednesday!
- Plate target cells in 6-well plate for infection on Thursday. Make sure to have an extra plate/well of uninfected or mock infected cells to use as a canary during selection. You can also plate your cells on Tuesday and allow two days to recover. I typically infect a plate that is 30-40% confluent. Keep in mind that you will be growing them for the rest of the week, and it would be optimal to split them on the following Monday just prior to beginning selection, the selection is more efficient this way.
- Feed S293T cells. Be careful-you are handling virus here!
Filter media from S293T cells through a 0.45 micron filter attached to a 10-mL syringe (or 5 mL if you have these). Add filtered media to target cells. Add ~8ug/mL polybrene and allow to incubate for four hours at 37C. Change media on target cells back to their normal growth media.
- Repeat same process as was done for Thursday. (double infection)
- Allow cells to grow over the weekend. On Monday, I expanded my cells from the 6-well plate on to a 10-cm plate and began selecting with puromycin (1ug/mL to start). Change media every two days as normal. Make sure you have a canary plate (an uninfected plate that you can select with drug and see how long it takes for uninfected cells to die off. I used a mock transfection well and carried it through the entire week so that I would have approximately the same number of cells on that plate as was on the others).