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

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04/09/11

 * &#x2713; 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

> 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&deg;C > Assay luc activity after 2 days


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