Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/06/03
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On Monday I started a 20 mL culture of K562 in antibiotic-free medium. Today I'll be checking cell density and planning out the transfection setup.
Measured at 300,000 cells/mL by hemacytometer.
3.0E5 cells/mL * 20 mL = 6.0E6 cells total
The plan is to collect 1 control (GFP) and 2 samples (KAH126-MV2) for each day of collection, and to collect on days 2 and 3 (24 hours & 48 hours post-transfection). This makes 6 samples total. I'll be using a 6-well plate for this experiment and transfecting using the 100 µL tips.
Cells will be resuspended in buffer at 2.0 x 107 cells/mL for transfection. Multiplied by 8 transfections (2 buffer), that's 2.0E7 cells/mL * 0.8 mL = 1.6E7 cells needed.
Number of cells per well: 0.4-1 x 106
Initial cell density after transfection and transferring to wells (in 2 mL plating medium) will be 2.0E7 cells/mL * 0.1 mL / 2.0 mL = 1.0E6 cells/mL
Amount of DNA per sample is 5-30 µg, not to exceed 10% of total volume of cells (100 µL * 0.1 = 10 µL). Both pMaxGFP and KAH126-MV2 are at concentrations of 2.0 µg/µL, so 10 µL per sample would be 20 µg of DNA per sample.