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

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==Summary==
==Summary==
Explain what you're doing today and why. Paragraph format, simple description so somebody reading the notebook has some orientation.<br><br>
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.<br><br>
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.<br><br>
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.<br><br>


==Goals==
==Goals==

Revision as of 13:16, 24 December 2014

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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

  • 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

Any significant data goes here. Thoughts on what happened, significance of findings, what to do next etc.
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