Altman:Protocols/Protein Purification/SF9 transfection: Difference between revisions
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: PROTOCOL FOR COUNTING CELLS: | : PROTOCOL FOR COUNTING CELLS: | ||
: 1) Get SF9 cells from the incubator shaker, and put in TC hood | : 1) Get SF9 cells from the incubator shaker, and put in TC hood | ||
: 2) Remove 10 | : 2) Remove 10 uL of the cells, being sure to shake the flask beforehand | ||
: 3) Dilute the cells 5x with 40 uL of Trypan blue | : 3) Dilute the cells 5x with 40 uL of Trypan blue | ||
: 4) Place 10 | : 4) Place 10 uL in a hemocytometer (from Shirley’s bench) | ||
: 5) Count the cells in 4 square grids | : 5) Count the cells in 4 square grids | ||
:: 1 grid = 10-4 mL | :: 1 grid = 10^-4 mL | ||
:: Cell Density = (mean # of cells per grid)*5*10^4 cells/mL | :: Cell Density = (mean # of cells per grid)*5*10^4 cells/mL | ||
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6. Dilute 10 ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube | 6. Dilute 10 ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube | ||
7. Dilute 150 | 7. Dilute 150 ul of ESCORT IV cationic lipids in 1.35 mL of unsupplemented media; mix by inverting tube | ||
8. Add diluted DNA dropwise to diluted lipids, mixing while adding | 8. Add diluted DNA dropwise to diluted lipids, mixing while adding |
Revision as of 10:31, 19 February 2013
Direct transfection of SF9 cells for small-scale protein purification
(Protocol is from DA, based on ZB, based on Novagen protocol)
- This protocol is for transfecting 15 mL cultures of SF9 cells at 1*10^6 cells/mL. 3 mL of DNA:lipid complexes are used for each 15 mL culture of cells. The protocol can be scaled up slightly by making a larger batch of DNA:lipid complexes (I have made up to 10 mL at a time) and splitting it among multiple 15 mL cultures in separate flasks.
1. Determine the cell density
- PROTOCOL FOR COUNTING CELLS:
- 1) Get SF9 cells from the incubator shaker, and put in TC hood
- 2) Remove 10 uL of the cells, being sure to shake the flask beforehand
- 3) Dilute the cells 5x with 40 uL of Trypan blue
- 4) Place 10 uL in a hemocytometer (from Shirley’s bench)
- 5) Count the cells in 4 square grids
- 1 grid = 10^-4 mL
- Cell Density = (mean # of cells per grid)*5*10^4 cells/mL
2. Determine the volume of cells required for the day
- Each transfection requires 15 mL of SF9 cells at 1*10^6 cells/mL, or 1.5*10^7 cells
3. Wash the cells by spinning 2 min at 500 rpm in a 15 mL conical in a Beckman TJ-6 centrifuge
4. Remove the supernatant, and re-suspend the pellet in unsupplemented SF 900 II media to a cell density of ~1.5*10^6 cells/mL (10 mL of media per transfection)
5. Replace cells in 28° shaker until the complexes are ready for transfection
6. Dilute 10 ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube
7. Dilute 150 ul of ESCORT IV cationic lipids in 1.35 mL of unsupplemented media; mix by inverting tube
8. Add diluted DNA dropwise to diluted lipids, mixing while adding
- Ensure complete mixing by inverting tube afterwards. Allow mixture to sit in the hood for at least 15’ to promote complex formation.
9. Meanwhile, retrieve and count cells; dilute the cells to a final density of 1.25 *10^6 cells/mL in unsupplemented media
10. Dispense 12 mL of cell culture into a plastic 125 mL culture flask
11. Add 3 mL DNA:lipid complexes to the cell culture.
12. Replace cell culture in 28° shaker; cells may be harvested in 48-72 hours