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.