Altman:Protocols/Protein Purification/SF9 transfection: Difference between revisions
David Altman (talk | contribs) (New page: {{Template:Altman}} <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> ==Direct transfection of SF9 cells for small-scale protein purification== (Protocol is from DA,...) |
David Altman (talk | contribs) No edit summary |
||
(4 intermediate revisions by the same user not shown) | |||
Line 10: | Line 10: | ||
1. Determine the cell density | 1. Determine the cell density | ||
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 | : 2) Remove 10 uL of the cells, being sure to shake the flask beforehand | ||
3) Dilute the cells 5x with | : 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 | |||
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 10 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 |
Latest revision as of 11:38, 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 10 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