I'll be optimizing the neon transfection system (Day 1) for BT-474 and BT-549 cultures.
- Prepare a 24-well plate containing 0.5 mL culture medium
- Culture cells in T-75 flasks until they reach 75-90% confluency. Wash, trypsinize, and resuspend in fresh medium. Spin down cells (200g, 5 min) and resuspend in 1 mL PBS.
- Take a 20 µL aliquot of the resuspended cells and measure cell density using flow cytometry. Calculate the total number of cells in your sample. Transfer 5x104-2x105 cells per sample (1.5x106-6x106 for 24 wells + 6 buffer) into a fresh tube and centrifuge. Resuspend in Resuspension Buffer R at a final concentration of 1.0x107 cells/mL (300 µL total).
- Add 1 µg of plasmid DNA per sample (30 µg total) to your cells. DNA should be at a concentration of 1-5 µg/µL.
- Set up a Neon Tube with 3 mL Electrolytic Buffer into the Neon Pipette Station.
- Press Optimization and load the optimization protocols to begin electroporation. See parameters listed on page 24 of the Neon Transfection System manual.
- After electroporation, transfer samples from the 10 µL Neon Tip into the prewarmed 0.5 mL culture medium.
- Rock the plate to ensure even distribution of cells, then incubate at 37°C