Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/16

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

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Summary 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 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. David Benjamin Nyer 18:36, 16 December 2014 (EST):