MDCK transfection Protocol

MDCK Effectene transfection protocol MDCK Cells must be seeded at least 5.5 hours before transfection.

Tranfection Reagent used : Effectene (Qiagen) 1) 0.25 ug DNA per construct in Buffer EC to final volume of 100 ul

2) Add [8 x Net amount of DNA used (ug) ] ul of Enhancer reagent

3) Vortex for 1 second

4) Incubate 2–5 minutes at RT

5) Add Effectene Transfection Reagent, 5 ul per Mattek dish (amount of DNA irrelevant) 6) Incubate the transfection mix for 5–10 min at RT. While incubation proceeds, replace medium in Mattek dishes with 1.5‐2.0 ml OptiMEM w/Glutamax (Invitrogen). 7) Vortex for 10 seconds after incubation and add OptiMEM to transfection mix. 8) Pipet up and down twice. 9) Add transfection mix to cells in Mattek Dish. 10) Incubate dishes at 37 deg C Cells express protein 6‐7 hours after transfection. Incubate overnight for maximal exprssion.

- Submitted by Nachiket Vartak