User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30

Pc-TF Genomics

Main project page Previous entry      Next entry

6/30/13 Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox PcTF transfection of SK-N-SH Gal4-EED/luc cells +dox Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range. 1.0 μg/mL dox 10-1 10-2 10-3 10-4 0 Addition of serial dilutions of dox to cells Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary. Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...   tube 1 2 3 4 5 6 1 mg/mL dox (μL) 8.0 --- --- --- --- --- mixture from prior tube (μL) --- 100 100 100 100 --- +growth med. (μL) 1000 900 900 900 900 --- Final [dox] (μg/mL) in 4 mL med. 1.0 10-1 10-2 10-3 10-4 0 Lipo transfection > Samples are for RT-PCR > Cells were 100% confluent > Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium > Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH126/MV2); 226 ng/μL; Note: no TetR in SK-N-SH, so expression will be constitutive Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo 1 KAH126/MV2 2 μg 8.8 μL +11.2 570 μL 2.5 μL 7.5 μL 2 KAH126/MV2 2 μg 8.8 μL +11.2 " " " 3 KAH126/MV2* 2 μg 8.8 μL +11.2 " " " 4 mock none 0 μL + 20.0 " " " 5 mock none 0 μL + 20.0 " " " 6 mock none 0 μL + 20.0 " " " (*Ran out of plasmid DNA) > Add 570 μL Opti-MEM to each 20 μL DNA sample > Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp > Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp > Add 600 μL complexes to each well (final volume of medium = 4 ml/well) > Grow cells at 37°C overnight Results (7/01/13) Checked RFP expression on the Nikon scope Expression is good. Expand cells in 10 cm plates Do TRIzol prep after ~10 days.