# Difference between revisions of "Altman:Protocols/Protein Purification/SF9 transfection"

Department of Physics, Willamette University

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## 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