Eccles:AJ siRNA txn

Transfecting siRNA into mammalian cells by using Lipofectamine 2000

This protocol is for use with cells seeded into a 24-well plate with siRNA at 100 nM final concentration in 200 uL/well. Scale volumes according to taste and need, and siRNA stock concentration (the volumes below are for 50 uM siRNA stock).

• lipofectamine 2000 (Li2k; Invitrogen).
• Opti-MEM (Invitrogen).
• siRNA (no more than 100 nM final concentration).
• sterile 1.5 mL tubes.

Transfection is done according to the manufacturer's instructions, but with final volume of 200 uL in 24-well plate, rather than 100 uL (I find 100 uL too little to easily cover bottom of 24-well, and the reagent volumes are on the small side to accurately pipette).

For one transfection i.e. one well:

1. Dilute appropriate volume of siRNA in Opti-MEM without serum (or other medium without serum) to final volume of 50 uL, and mix gently and well by pipetting.
   • 0.4 uL (50 uM) siRNA
   • 49.6 uL Opti-MEM
2. Mix li2k gently before use, then, in a separate tube, combine li2k with Opti-MEM, and mix well by pipetting.
   • 1 uL li2k
   • 49 uL OptiMEM
3. Allow mixed li2k/OptiMEM to incubate at RT for 5 min.
4. Combine siRNA mix with li2k mix, mix well by pipetting, and incubate at RT for 20 min (this is for the the siRNA to complex with the transfection reagent).
5. Add 100 uL (equal volume) of Opti-MEM, and mix well by pipetting. Final volume is now 200 uL with siRNA at 100 nM.
6. remove normal growth media from cells, wash 2 x with OptiMEM.
7. Add 200 uL transfection mix from (5).
8. 4-6 hours post-tranfection, add equal volume (200uL) normal growth media (MEM-alpha for NZM cells) or OptiMEM containing 10% FCS ( to achieve 5% FCS).
9. For replicate transfections, make up master mixes, for example:

x1 x4 siRNA (50 uM) 0.4 1.6 Opti-MEM 49.6 198.4

AJ April 2007.