User:Karmella Haynes/Notebook/miR Trigger/2011/01/23

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01/23/11

  • ✓ Transfection: test RFP-repressors
  • ✓ Culture: thaw JDS33-47



Lipo transfection
> RFP-Repressors with new BGH polyA (from Mara) cloned in forward and reverse orientations in MV8/zeo vector (cloned by Andrew)
> Vector has BGH polyA in fwd orientation; RFP expression from reverse clones will show that BGH polyA BioBrick works

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH176/MV8 fwd 2 μg 2.7 μL 4 μL 500 μL
2 KAH176/MV8 rev " 5.1 μL " "
3 KAH177/MV8 fwd " 3.5 μL " "
4 KAH177/MV8 rev " 5.0 μL " "
5 KAH178/MV8 fwd " 3.7 μL " "
6 KAH178/MV8 rev " 5.3 μL " "

> Change medium to ab-free (3.5 mL)
> 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