Haynes Lab:Notebook/Engineering PC-TFs/2013/10/23

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October 23, 2013 Transfection of KAH126 Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total) 1-2 KAH126 1.5 μg 23 μL > 20 μL 0μL 2.5μL 7.5 μL 570 μL Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: Label sterile microfuge (1.5 ml) tubes. Add 570 μL Opti-MEM to each 20 μL DNA sample. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells. Incubate cells at 37°C in a CO2 incubator (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium. Cell Culture of KAH126 Grow liquid culture of KAH126 Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp. Use sterile pipette tip to touch bacterial streak and put the tip into LB medium. Grow the cultures for 10-12 hrs.