Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/24

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines

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Summary The second transfection attempt is also showing extremely low levels of PcTF-mCherry protein in the U2OS cell line (fluorescence microscopy images not shown here). Today being Christmas Eve, I'm going to wait until next week before making another attempt. Today, I'll be passaging U2OS cells at 1:20 dilution and checking on SKNSH cell lines. Next week, set up a 6 or 12 well plate with very dilute U2OS cells and perform daily cell counts using flow cytometry to determine how quickly growth is occurring and when to transfect. Ideally, transfection occurs with 1.25E5-2.5E5 cells, but my suspicion is that, due to the rapid growth of U2OS, we're using more cells than necessary, resulting in low yields. Also to do next week: thaw the U2OS dox-inducible PcTF-mCherry cell line and begin growth, and set up a 6 well plate for SKNSH cell line transfection with PcTF-mCherry plasmid. Goals Passage U2OS cells from PS-2 to PS-3 Protocol splitting cells Materials 70% ethanol spray bottle 1x Phosphate-buffered saline (PBS) Trypsin-EDTA buffer/ media Complete cell culture medium (e.g., 10% FBS, 1% pen-strep) Sterile tissue culture-treated vent-capped T-75 flask, one per new culture Pipette tips, etc. 100% confluent (dense) cell culture (check confluency under the light microscope) Notes Observations, measurements, placeholder info etc. go here Results/Conclusions U2OS cells in passage PS-3 are in a T-75 flask in the upper incubator, third shelf from the top. David Benjamin Nyer 15:18, 24 December 2014 (EST):