# 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.