Haynes Lab:Notebook/Engineering PC-TFs/2015/02/14

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Summary

Transfection

  1. Brought 2.0 μg total plasmid DNA up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. 10μL BL01v(202ng/μL) + 10μL dH2O, 6μLBL09 (333ng/μL) + 14μL dH2O.
  2. Took the DNA samples to the tissue culture room.
  3. In the tissue culture room, warmed GAL4EED-Luc antibiotic-free growth medium at 37°C.
  4. Warmed Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
  5. Prepared DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubated for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubated for 30 minutes at room temperature.
  6. Removed the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet.
  7. Aspirated off the old antibiotic-containing medium in the wells.
  8. Washed with 1mL DPBS.
  9. Added 4mL of warm antibiotic-free growth medium to each well.
  10. Added the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  11. Incubated cells at 37°C in a CO2 incubator