User:Karmella Haynes/Notebook/miR Trigger/2009/12/17

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12/17/09

  • ✓ Transfection: RFP reporter into JDS37-43 (luc miRNA-YFP)

Transfections
> U2OS JDS37-43 (luc miRNA-YFP) cells + RFP-luc target reporters
> Use Lipofectamine, 6-well format

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH93/MV2 2 μg 4.4 μL 4 μL 500 μL
2 KAH94/MV2 " 4.8 μL " "
3 KAH95/MV2 " 8.7 μL " "
4 KAH10/pcN " 4.5 μL " "
5 no DNA --- --- " "
6 untreated --- --- --- "

Day 1 (today)
> 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.
> Remove 0.5 mL growth medium from each well before adding transf. mix. Add 500 μL complexes to each well (total vol./well = 4 mL); Grow cells at 37°C
> After 5 hours, change medium (ab-free)

Day 2 (12/18/09)
> Transfection (DNA+Lipo) was slightly toxic, no need to split cells; change medium to 15μg/mL puro(morning)
> Do microscopy to find RFP+ cells (record positions using Metamorph multi-position staging)
> Add 1 μg/mL doxycycline

Day 3
> Microscopy: record RFP vs. YFP expression at set stage positions

Day 4
> Repeat Day 3