User:Karmella Haynes/Notebook/PcTF Genomics/2014/10/08

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10/08/14

  • "Spinoculation" transfection expriment



"Spinoculation" transfection expriment

  • Try centrifuge spin approach to increase transfection efficiency
  • See notes from
  • Use U2OS cells


Samples

  1. KAH126/MV2, spin
  2. mock, spin
  3. KAH126/MV2 conventional
  4. mock conventional


Spinoculation

  • Set up Lipo-DNA and mock complexes for 6-well plates (standard Lipo LTX protocol): 2000 μg/ 20 μL DNA, 570 μL Opti-MEM, 2.5 μL PLUS, 7.5 μL Lipofectamine LTX
  • Harvest U2OS cells from T-75 flask (~1.0E6 cells/ mL)
  • Transfer ~5.0E5 cells into sterile 15 mL conicals. Fill up to final vol. of 3.4 mL with growth medium
  • Add 0.6 mL Lipo-DNA or mock complexes. Swirl to mix.
  • Spin at 3000 rpm (800 - 1000 xg) at room temp for 25 min. Note: centrifuge is IEC Centra-CL2 with rotor 215.
  • Transfer most of supernatant into 6-well plate wells
  • Flick to disrupt the pelleted cells. Use 1 mL of growth medium to transfer cells to wells.

Conventional

  • Remove 0.5 mL growth medium from each well
  • Drop-wise, add Lipo-DNA or mock complexes to wells


Grow all cells overnight at 37°C/5% CO2. Check for RFP expression tomorrow



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