SwitchGear Genomics © SwitchGear Genomics

TRANSFECTION PROTOCOL FOR ADHERENT CELLS

DAY 1
Seed Cells for Transfection
  • Seed cells in a white tissue culture plate to yield ~40-80% confluence 24 hours later
    • Seed in a clear-bottom plate in parallel to assess confluence
    • 2,000-5,000 cells per well (96-well format) and 1,000-3,000 cells per well (384-well format) typically yields target confluency
  • Seed in 100uL total volume for 96-well plate, 30uL total volume for 384-well plate
DAY 2
Prepare Plasmid DNAs
  • Thaw at room temperature
  • Centrifuge to remove condensation from lid
  • Carefully remove foil plate tape
Transfections
  • Conduct at least 3 replicate wells for each transfection
  • Make a master mix, for the entire experiment, of Fugene + OptiMEM to add to each construct (add Fugene to OptiMEM without touching sides of tube)
  • For each construct, make a master mix of DNA, Fugene, OptiMEM (make at least 0.5 extra aliquots)
We recommend the use of at least 50ng of plasmid DNA per well for 96-well experiments and 30ng of plasmid DNA per well for 384-well experiments

Per well
(96-well format)
Per well
(384-well format)
  
1.67uL
1.00uL
 Experimental Plasmid (30ng/uL)
0.30uL
0.12uL
 Fugene-6 Transfection reagent
3.03uL
1.88uL
 Opti-MEM (serum free media)
5.00uL
3.00uL
 TOTAL
  • Mix DNA, Fugene, OptiMEM combination well.  Let sit at room temperature for 30-60 minutes.
  • Gently drip 5uL or 3uL (96- or 384-well formats) onto seeded cells
  • Shake gently, cover with lid or breathable sealing tape
  • Put back in incubator for 24-48 hours (+/- stimulus of interest)
Cells that transfect poorly may require the use of more DNA and/or longer incubation times.  We recommend optimizing conditions for each cell line before beginning large-scale experiments.

DAY 3
Assay
  • Remove plate from incubator and bring to room temperature
  • Add 100uL (96-well format) or 30uL (384-well format) Promega Steady-Glo Luciferase Assay Reagent, cover tightly with foil tape
  • Shake 15 minutes at room temp
  • Centrifuge, gently, to bring liquid to bottom of wells
  • Read in a plate luminometer – 2 seconds per well
Example catalog numbers for reagents
Item Vendor Catalog Number
White Tissue Culture Plates (96-well solid bottom) E&K Scientific EK-25073
  VWR 62402-976
Clear Tissue Culture Plates (96-well) E&K Scientific EK-25185
  VWR 62406-060
White Tissue Culture Plates (384-well solid bottom) E&K Scientific EK-30073
  VWR 62409-072
Clear Tissue Culture Plates (384-well) E&K Scientific EK-30182
  VWR 62409-074
Fugene-6 Transfection Reagent Roche 11814443001
Opti-MEM Invitrogen 31985-070
Steady-Glo Luciferase Assay Reagent Promega E2510, E2520
Foil Plate Sealing Tape E&K Scientific T592100
Breathable Plate Sealing Tape E&K Scientific T896100-S
  VWR 47749-926
Plate Luminometer Molecular Devices LMaxII-384