Nucleofections

From VLP

Order si’s from Dharmacon at 2nmol x 4 pack

Dilute siRNA in 40µl siRNA resuspension buffer (in fridge by Shiying’s bench)

Use 1nmol to test si then work down or up depending on efficiency

Get Amaxa nucleofection solution from 4ºC walk-in and add supplement. Label with date and use within 3 months.

Nucleofection kit with cuvettes are by Cari’s bench.

Control siRNA in common space in freezer in chemical room.

1. Set up 3 sets of eppi-tubes per siRNA: 1-cells; 2-siRNA; 3-recovery cell suspension

2. Aliquot siRNA into eppi-tubes.

3. Prepare p150 plates that nucleofected cells will go into (one plate per nucleofection).

4. Split cells (will need 2-4 x 106 cells per nucleofection) and resuspend in 1ml 50/50 per nucleofection (e.g. if 10 nucleofections, resuspend in 10ml)

5. Count cells and aliquot up to 1ml into first set of eppi-tubes of desired amount of cells (2-4 x 106)

6. Spin down cells in eppi-tubes for 5 min at 2000rpm. Aspirate media and spin down again for 1 min 2000rpm—pipet out remaining media.

7. Resuspend cell pellets one at a time in 100µl nucleofection solution (cells do not like being in this solution—pelleted cells will be ok).

8. Add cell suspension to siRNA eppi-tube and mix.

9. Add cell suspension plus siRNA mix to cuvette, insert cuvette into Amaxa machine and use high viability or high efficiency program. Press enter, cuvette will move into machine for 30 seconds and return to original position.

10. Carefully add 500µl 50/50 media to cuvette. Use plastic pipet to remove cell suspension from cuvette. Place into 3rd set of eppi-tubes and move to 37º incubator.

11. Once all nucleofections complete, remove cells from incubator and plate into p150 with 50/50

12. Let cells recover for 24h and proceed with analysis.

Testing siRNA:
 * Set up enough for subconfluent for 3-4 days and differntiation 3-4 days—will need ~3-4 x 106 cells for single set, 6-7 x 106 for RNA and protein.
 * For subconfluent cells, seed 400K cells for 48h time point then ½ amount thereafter (e.g. 200K for 72h, 100K for 96h…) in p60 + 4ml 50/50 media.
 * For differentiation: seed 400-450K cells per well in 12-well plate + 3ml 50/50 media. On first day, change entire amount of media, every day thereafter, change half media.