Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/19

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Summary

NOTE: Today's procedure is a repeat of the experiment on 12/16/2014, using a higher-concentration plasmid DNA source.

In order to introduce a new gene into the mammalian cancer cells, we need to make the cells competent and present them with a prepared plasmid containing our gene of interest along with a selectable marker. Lipofectamine is a tranfection reagent that produces positively charged liposomes that envelop the plasmids to be introduced to the cells and encourage passage through the negatively charged cell membrane into the cells.

Goals

  • Begin transfection of U2OS cells in 6-well plate

Protocol

mammalian cell transfection with liopfectamine

Materials

  • Lipofectamine LTX
  • Opti-MEM I Reduced serum medium
  • Antibiotic-free growth medium (no pen/strep or other antibiotics)
  • Cells
  • Pipette tips

Notes

To get your plasmid to the appropriate concentration, add 2.0μg to each 1.5mL microcentrifuge tube and then add nuclease-free water to bring the total volume to 20μL.

To make the transfection solution, in a 1.5mL microcentrifuge tube add:

  • 20μL DNA in H2O
  • 570μL Opti-MEM solution
  • 2.5μL PLUS reagent
  • wait 5 minutes at room temp.
  • 7.5μL Lipofectamine LTX
  • wait 30 minutes at room temp.
  • begin transfection

Results/Conclusions

Three wells were treated with DNA, one was treated with water (negative control). Well plate is in the upper incubator, middle shelf.
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