ure. Mix by pipetting up and down 5 times, or by vortexing for 10 s. 6. Incubate the samples for 10 min at room temperature (1525C) to allow transfectioncomplex formation. 7. While complex formation takes place, gently aspirate the growth medium from the plate, and carefully wash cells once with sterile PBS using 1.52 times the volume of medium used for cell seeding.
IMPORTANT:
Do not allow the cells to become dry. Minimize the time they are without medium. 8. Add 900 l cell growth medium without serum or antibiotics to the tube containing the transfection complexes. Mix by pipetting up and down twice, then immediately add the transfection complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.
IMPORTANT:
Use medium without serum or antibiotics to avoid RNase contamination. 9. Incubate cells with the transfection complexes for 3 h under their normal growth conditions.
Note:
If no cytotoxic effects are observed, the incubation time can be increased up to 4 h. 10. Remove the complexes from the cells, wash cells once with PBS, then add 2 ml fresh medium containing serum and antibiotics to the cells. 11. Incubate cells under their normal growth conditions to allow for protein expressio
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