Eccles:AJ siRNA txn: Difference between revisions
No edit summary |
mNo edit summary |
||
Line 6: | Line 6: | ||
==Reagents== | ==Reagents== | ||
*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=541& Lipofectamine 2000] ( | *[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=541& Lipofectamine 2000] (Li2K; Invitrogen). | ||
*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=31 Opti-MEM] (Invitrogen). | *[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=31 Opti-MEM] (Invitrogen). | ||
*siRNA (no more than 100 nM final concentration, titrated according to specific siRNA and cell line). | *siRNA (no more than 100 nM final concentration, titrated according to specific siRNA and cell line). |
Revision as of 02:22, 21 April 2007
Back to Eccles Lab > DGG Protocols
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).
Reagents
- Lipofectamine 2000 (Li2K; Invitrogen).
- Opti-MEM (Invitrogen).
- siRNA (no more than 100 nM final concentration, titrated according to specific siRNA and cell line).
- sterile 1.5 mL tubes.
Transfection is done according to the manufacturer's instructions (PDF), 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).
Protocol
For one transfection i.e. one well:
- 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
- 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
- Allow mixed Li2K/OptiMEM to incubate at RT for 5 min.
- 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).
- Add 100 uL (equal volume) of Opti-MEM, and mix well by pipetting. Final volume is now 200 uL with siRNA at 100 nM.
- remove normal growth media from cells, wash 2 x with 500 uL OptiMEM.
- Add 200 uL transfection mix from (5).
- 4-6 hours post-tranfection, add equal volume (200uL) normal growth media (MEM-alpha for NZM cells) or OptiMEM containing 2x the normal FCS percentage for your particular cell type (to achieve 1x FCS).
- For replicate transfections, make up master mixes.
Example
Three different siRNAs - scrambled negative control, GAPDH positive control, target gene - will be transfected into cells seeded at two different densities in duplicate => 3 siRNAs x 2 duplicates => 6 wells/cell density x 2 cell densitys = 12 wells in total => 12 transfections in total (don't forget to factor-in tranfection reagent-only controls, if you're doing them - no siRNA tubes, but additional Li2K wells).
Make up the siRNA mix for each respective siRNA in a separate tube - there are 4 tranfections (wells) for each siRNA (n=2 density 1 + n=2 density 2 => 4 wells/siRNA):
x1 (uL) | x4 (uL) | |
---|---|---|
siRNA (50 uM) | 0.4 | 1.6 |
Opti-MEM | 49.6 | 198.4 |
Total | 50 | 200 |
The siRNAs are prepared in three sperate tubes, but the Li2K can be made up in one tube containing the appropriate amount of Li2K and Opti-MEM for all the transfections, i.e. 12 (again, don't forget to factor-in tranfection reagent-only controls, if you're doing them - no siRNA tubes, but additional Li2K wells):
x1 (uL) | x12 (uL) | |
---|---|---|
Li2K | 1 | 12 |
Opti-MEM | 49 | 588 |
Total | 50 | 600 |
AJ April 2007.