User:Karmella Haynes/Notebook/miR Trigger/2009/12/29

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12/29/09

  • ✓ Transfection 1: RFP-reporters KAH93-95 for time-lapse microscopy
  • ✓ Transfection 2: RFP-reporters KAH93-95 for RNA(RT-PCR)



Transfection 1
> KAH93-95, 108/MV2 + JDS33-47 U2OS
> 12-well format, Fugene

Wells Plasmid DNA Volume Fugene Opti-MEM
Col.1 (1-3) KAH93/MV2 400 ng 1.9 μL 3.6 μL 46.4 μL
Col.2 (4-6) KAH94/MV2 " 2.4 μL " "
Col.3 (7-9) KAH95/MV2 " 2.3 μL " "
Col.4 (10-12) KAH108/MV2 " 0.9 μL " "

> Master mixes (4, x3): 10.8 μL Fugene + 139.2 μL Opti-MEM --> R.T/ 5 min.
> Add 3x vol DNA --> R.T./ 20 min.
> Add 50 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight

Day 2
> Change to CO2 independent medium (L-15, no phenol red, 10% T.S. FBS, 1% P/S)
> Mark anchor point on well 1
> Set up plate under scope; secure plate to ensure no shifting; set to 34°C (Note: heater is +3-4°C off)
> Find RFP+ cells and set staging
> Add 1 μg/mL Dox; record images every 2-4 hours


Transfection 2
> KAH93-95/MV2 + JDS33-47 U2OS
> 6-well format, Lipofectamine 2000

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH93/MV2 2 μg 9.4 μL 4 μL 500 μL
2 KAH93/MV2(Dox) " 9.4 μL " "
3 KAH94/MV2 " 12.0 μL " "
4 KAH95/MV2(Dox) " 12.0 μL " "
5 KAH95/MV2 " 11.3 μL " "
6 KAH95/MV2(Dox) " 11.3 μL " "

> 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.
> Culture vol. = 3.5, no need to remove 500 mL. Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)

Day 2
> Add 1μg/mL Dox to "Dox" wells.

Day 3
> RNA preps/ cDNA synthesis