To begin, aliquot 200 microliters of NBIL media into 1.5 milliliter tubes. Add 26 microliters of the optimum transfection reagent mixture to the DNA optimum mixture. Pipe at the reagents vigorously approximately 10 times.
Transfer the required volume of mPSC suspensions to the 200 microliters of NBIL tubes. Then add lipofectamine 2000 and DNA mixture to the tubes and mix well by pipetting. After five minutes, centrifuge the mixture at 300 G for five minutes and remove the supernatant, leaving approximately 50 microliters in the tube.
Re suspend the pellet in 100 microliters of NBIL media. Transfer the cell suspension to a gelatin-coated 24-well plate and place plate in the incubator. Transfer the required volume of mPSC suspension to seed 25, 000 cells per well of a gelatin coated 24-well plate and place the plate in the incubator.
One hour prior to transfection. Replace the media from the wells with 0.5 milliliters of NBIL media. After incubation, dropwise, add lipofectamine 2000 and DNA mixture to the wells.
Place the plate in the incubator for 48 hours.
