Difference between revisions of "Lipofectamine Mediated Knockdown"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (rewrote according to lipofectamine instructions) |
Davebridges (Talk | contribs) m (fixed typo pmoles should be nmoles) |
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==Required Amounts== | ==Required Amounts== | ||
*Calculate RNA to transfect (typically 40 pmol per well) | *Calculate RNA to transfect (typically 40 pmol per well) | ||
| − | *Lipofectamine(in uL) = RNA(in | + | *Lipofectamine(in uL) = RNA(in nmoles) * 50 |
*OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50 | *OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50 | ||
Latest revision as of 16:39, 18 December 2009
Materials
- Cells in a 12 well dish
- Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
Required Amounts
- Calculate RNA to transfect (typically 40 pmol per well)
- Lipofectamine(in uL) = RNA(in nmoles) * 50
- OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50
Protocol (6 well dish)
- Plate cells the day before from a confluent dish diluted to 1:12 into DMEM/FBS with no PSG
- For each well prepare, scalin up as required:
- 100uL OptiMEM plus 40 pmoles of RNA.
- 100uL OptiMEM plus 2 uL of Lipofectamine.
- Incubate ~5 minutes at room temperature.
- Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine (should be 200uL/well).
- Incubate ~20 min at room temperature.
- Add DNA/Lipofectamine complexes (~200 uL) to the 1 mL of media on the cells
- Gently rock plate to mix
- After ~4h re-feed cells with normal media.
Protocol adapted from Product Manual
