User:Karmella Haynes/Notebook/Polycomb project/2011/04/09

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04/09/11

  • ✓ Transfection: Pc-TF's + luc-repressed HEK's (2 day w/o)



Fugene transfection

  1. KAH160: human Pc-TF
  2. KAH165: human PCD
  3. KAH161: fly Pc-TF
  4. KAH167: fly PCD
  5. KAH162: fish Pc-TF
  6. KAH166: fish PCD
  7. KAH170: deleted PCD
  8. mock (Fugene)


--> Plate 1: no dox
--> Plate 2: Dox-treated cells (2 days, wash-out day 2)
--> Plate 3: Dox-treated cells (4 days, wash-out day 2)
--> Plate cells: Collect cells from 2 10cm plates, resuspend in 25 mL medium, aliquot 1 mL per well in 24-well plate


Wells Plasmid DNA Volume Fugene Opti-MEM
1-3 KAH160/MV1 100, 200, 400 ng 1.2 μL = 400 ng 1.8 μL 18.2 μL
4-6 KAH165/MV1 " 0.8 μL = 400 ng " "
7-9 KAH161/MV1 " 1.0 μL = 400 ng " "
10-12 KAH167/MV1 " 1.0 μL = 400 ng " "
13-15 KAH162/MV1 " 1.1 μL = 400 ng " "
16-18 KAH166/MV1 " 0.9 μL = 400 ng " "
19-21 KAH170/MV1 " 1.0 μL = 400 ng " "
22-24 mock " 0 " "

> Add DNA to stdH2O for a total volume of 5 μL (3x master mix = 15 μL)
--> Serial dilutions: start w/ 6x DNA + dH2O = 30 μL --> Use 15 μL for serial dilutions
> Fugene master mixes (x3, 24 total): 5.4 μL Fugene + 54.6 μL Opti-MEM --> R.T/ 5 min.
> Add 15 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days