PEI Mediated Plasmid Transfection

This protocol is modified from the Addgene website protocol

Materials

  • Cells to transfect, generally growing in log phase at ~90% confluence
  • Plasmids to transfect. Generally use 500 ng/well of a 6 well plate
  • OptiMEM
  • Chloroquine stocks (25 mM stocks). See Preparation of Chloroquine Stocks.
  • PEI (1 mg/mL stocks). See Preparation of PEI Stocks.
  • DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG


Transfection Procedure

  • The morning of the transfection day, replace the media with fresh DMEM without PSG and containing 10 uL of 25 mM chloroquine (optional). Wait ~5h before going onto the next step.
  • Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well.
  • Add 3 uL PEI per ug of DNA in OptiMEM. The total volume should be 100 uL per well. if transfecting multiple contstructs, make enough of this solution for all transfections.
  • Gently add the PEI solution dropwise into the DNA solution (adding 100 uL to each 100 uL/well volume).
  • Incubate at room temperature for 15-20min.
  • Add transfection mixture slowly to the cells.
  • Incubate overnight. The next day carefully replace with media containing PSG and/or treat as needed.