User:David Benjamin Nyer/Notebook/PcTF breast cancer/2015/10/28

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Summary

I'll be optimizing the neon transfection system (Day 1) for BT-474 and BT-549 cultures.

Methods

  1. Prepare a 24-well plate containing 0.5 mL culture medium
  2. 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.
  3. 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).
  4. 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.
  5. Set up a Neon Tube with 3 mL Electrolytic Buffer into the Neon Pipette Station.
  6. Press Optimization and load the optimization protocols to begin electroporation. See parameters listed on page 24 of the Neon Transfection System manual.
  7. After electroporation, transfer samples from the 10 µL Neon Tip into the prewarmed 0.5 mL culture medium.
  8. Rock the plate to ensure even distribution of cells, then incubate at 37°C



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