1. Calculate the volume of DNA you are adding to each well based on the concentrations of the midiprepped DNA and the mass (ng) of each plasmid you want to add. Total DNA transfected per well should be 1ug.
  2. Label a DNA dilution tube (2mL eppendorf) for each well you are planning to transfect. These tubes will have a total volume of 50uL.
  3. Based on the volumes of calculated in Step 1, add enough volume of DMEM to each dilution tube such that once you add your DNA you will have a total volume of 50uL.
    i.e. Volume of DMEM = 50uL - Volume of DNA
  4. Add the volume of DNA calculated in Step 1 to each DNA dilution tube. 
  5. Add 50uL of DMEM to new 2mL eppendorf tubes (same number of tubes as DNA dilution tubes).
  6. Add to each new tube 2uL of Lipofectamine 2000
  7. Incubate for 5-10 minutes.
  8. Add 50 uL of the Lipofectamine+DMEM mix to each of the DNA dilution tubes.
  9. Incubate for 30 minutes. 
  10. Transfer 100uL of each DNA+Lipofectamine+DMEM mix to its corresponding well. 
  11. Put transfected plate back into 37C incubator. 
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