2 Cell Modification

2.1 siRNA Transfection

This protocol is adapted from the ambion Ambion Life Technologies protocol (Protocol Pub. No. MAN0007836 Rev. 1.0)

This protocol is for 6 well plates. For reagent volumes for smaller well form factors, see here

2.1.1 siRNA Resuspension

  1. Briefly centrifuge the tube to ensure dried siRNA is at the bottom of the tube
  2. Dilute siRNA down to \(100 \mu M\) \((100 \frac{pmoles}{\mu L})\) using nuclease-free water
  3. Aliquot out some of the siRNA

Each well of a 6-well plate takes \(3 \mu L\) of siRNA. Therefore, aliquoting \(12 \mu L\) per tube is good enough for triplicates, plus a little extra for the ‘angel’s share’

  1. Store at \(\le 20 ^\circ C\)

2.1.2 Performing Transfection

  1. Split your cells and dilute them with media to \(200,000\ cells/mL\). You will need \(2mL/well\).
  2. In 1.5mL eppendorf, prepare the following per well:
Volume Ref #
Opti-MEM Medium 150uL 31985062
Lipofectamine RNAiMAX Reagent 9uL 13778150
  1. In another 1.5mL eppendorf, prepare the following per well:
Volume Ref #
Opti-MEM Medium 150uL 31985062
siRNA (10 uM) 3uL (30pmol) varies
  1. Pipette to mix each tube

  2. Mix siRNA and lipofectamine together in a 1:1 ratio (for a single well, this would be \(150 \mu L\) of each together. You can simply add the siRNA solution to your lipofectamine tube)

  3. Wait \(5\ minutes\). During this time, dispense \(2mL\) of your cell suspension in each well of a 6 well plate.

  4. Add \(250 \mu L\) of mixture to each well.

  5. Gently swirl and rock the plate to ensure even mixing of cells and siRNA particles

  6. Incubate for \(3\ days\). There is no need to change media.