• My NEB
  • Print
  • PDF
  • TransPass D2 Protocol 2: Transfection in the absence of serum

    Introduction

    The amounts below are given for a 12-well plate format. Use Table 2 to adjust the reagent volumes for other plate sizes.

    Protocol

    1. Plate cells so they reach a confluence of 60-90% at the time of transfection.
    2. For each transfection well, add 1.5 µg of plasmid DNA into 0.6 ml serum-free high glucose DMEM in a microcentrifuge tube.
    3. Gently mix TransPass D2 Transfection Reagent tube before pipetting. Do not vortex.
    4. Add 1.5-3 µl TransPass D2 Transfection Reagent per tube and mix well by flicking the tube. Incubate at room temperature for 20-30 minutes to form the transfection complexes.
    5. Wash cells once with serum-free medium.
    6. Aspirate the culture medium from the cells and immediately replace with the transfection mixture. Rock the plate gently in order to evenly disperse the complex mixture.
    7. Return the plate to the incubator and incubate cells for 2-3 hours.
    8. Replace transfection medium with complete growth medium containing serum and incubate for 24-72 hours before assaying.
    9. Replace medium 24 hours post-transfection or as needed to maintain healthy cells.