Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/12

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RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Main project page
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Summary

Today is the 5th day after transfection of the SKNSH cells, so I'm going to take a look at them under the fluorescence microscope and if there is still significant fluorescence, harvest them for RNA sequencing/quantification and run them on the flow cytometer for cell counting and fluorescence quantification.

I'll also be expanding the KAH126/U2OS culture, creating an extra flask for Dr. Haynes. These cells are now ready to be induced with doxycycline, switching on the PcTF-RFP gene.

Lastly, some lab maintenance as I make more LB ampicillin selection plates for growing transformed E. coli and making more TAE buffer, as well as cleaning up the gel station.



Goals

  • Place main goals for the day here



Protocol

example protocol



Materials

  • List materials here, to be gathered before the experiment
  • Consumables such as media, antibiotics, DNA, kits etc.



Notes

Observations, measurements, placeholder info etc. go here



Results/Conclusions

Fluorescence microscopy images of SKNSH cells transfected with PcTF-RFP, 5 days post transfection:

Well 1


Well 2

Well 3

Appears to be the same phenomenon that was occurring with transfected U2OS cells - red fluorescence is localized primarily in dead cells not adherent to the bottom of the vessel. Based on this, I'm going to hold off on collecting RNA and flow cytometry data from the SKNSH cells.



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