To begin, take cultured HEK293T from the incubator. Aspirate out the existing culture media from each well, and add two milliliters of fresh media per well. For transfecting the cells with split R-loop plasmid DNA, mix the DNA and PEI in 200 microliters of DMEM in a 1.7-milliliter microcentrifuge tube for each well.
Quickly vortex the DNA and PEI at level eight for approximately two seconds to ensure they mix well to form a complex. Then spin it down briefly in a mini centrifuge. After incubation, drop the mixture onto the surface of the culture media in the six well plate containing cells.
Tap the plate gently to evenly distribute the mixture, and incubate it in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for six hours. Afterward, remove the six well plate from the incubator, aspirate out the media, and wash each well with two milliliters of DPBS. Once the DPBS is removed, add 350 microliters of 0.05%trypsin-EDTA phenol red.
At the end of the incubation, add 1.7 milliliters of media to each well to stop the trypsin reaction. Now, transfer the cells along with media to a 50-milliliter tube, and spin at 300 G for five minutes in a tabletop centrifuge. Aspirate the media and resuspend the cell pellet with one milliliter of fresh media.
To seed the cells in a poly-D-lysine coated 96 well plate, add the necessary volume of cells in a 50-milliliter tube along with the appropriate volume of media. Using a multichannel pipette, dispense 100 microliters of the diluted cell suspension into each of the 96 wells. Incubate the cells for 18 hours at 37 degrees Celsius with 5%carbon dioxide.
