User:Karmella Haynes/Notebook/miR Trigger/2010/02/17

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02/17/10

  • ✓ Transfection: for RFP-repressor knock-down experiment



Lipo transfection
> Wells 1-9: JDS33-47 cells; Wells 10-12: U2OS (Flp-in T-REx) cells

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH94/MV2 2 μg 6.2 μL 4 μL 500 μL
2 KAH94/MV2(24hr dox) " 6.2 μL " "
3 KAH94/MV2(48hr dox) " 6.2 μL " "
4 5xluc-/MV2 " 4.0 μL " "
5 5xluc-/MV2(24hr dox) " 4.0 μL " "
6 5xluc-/MV2(48hr dox) " 4.0 μL " "
7 no DNA --- --- " "
8 no DNA(24hr dox) --- --- " "
9 no DNA(48hr dox) --- --- " "
10 KAH94/MV2 2 μg 6.2 μL " "
11 5xluc-/MV2 " 4.0 μL " "
12 no DNA --- --- " "

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Add 500 μL complexes to each well (4 ml final vol.); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Wells 3, 6, 9 (48hr dox): Add dox, final = 1 μg/mL


Day 2
> Wells 2, 5, 8 (24hr dox): Add dox, final = 1 μg/mL


Day 3
> Flow cytometry analysis of RFP and YFP expression