How to transfect HEK 293T cells

Open the biosafety cabinet sash to proper height and activate the blower. Spray down all racks, bottles, and hands with ethanol to maintain a sterile environment for working with human cells.

Set up two labeled tubes (Red and Yellow). Add 50 microliters of optimized medium (OMI) to each tube, then add DNA and p3000 reagent to the Yellow tube and lipofectamine to the Red tube.

Add one microgram of DNA to the appropriate tube and gently tap to settle. Wait 3-5 minutes for reagents to activate, then combine both tubes by pipetting 60 microliters and mix gently. Wait an additional 10 minutes.

Examine cells under the microscope to verify they are at 75-90% confluency. Only proceed with transfection if cells have sufficient coverage for successful DNA uptake.

Bring prepared cell dish to the biosafety cabinet, spray down all materials with ethanol, and place the cells inside the cabinet ready for DNA addition. Remember to spray hands when re-entering the hood.

Pipette the DNA-lipofectamine complex slowly into the cell culture wells in small drips distributed across the plate. Gently swirl if desired to distribute the liposomes evenly throughout the wells.

Transfer the culture plate to a CO2 incubator set to maintain proper temperature and carbon dioxide concentration. Seal the incubator securely and allow cells to incubate for 24-48 hours to express the inserted protein.