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

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

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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 Transfected cells are in the upper incubator, third shelf from the top.