Haynes Lab:Notebook/Jan/2015/08/24

Project name

Main project page Previous entry      Next entry

Transfection of U2OS Cells with BD004/BD006 Culture Name: U2OS Name of Plasmid: BD006/MV2 and BD004/MV2 Benchling Link for Plasmid 1: BD006/Chromatin Sensor 1 Benchling Link for Plasmid 1: BD004/Chromatin Sensor 2 Name of Kit Used: Lipofectamine LTX Protocol: Transfection with Lipofectamine Protocol 1 day before transfection: Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection. NOTE: if you are using a cell line that does not adhere strongly to the plate (eg, HEK cells), seed the cultures using antibiotic-free medium the day before to avoid having to replace medium the day of transfection. Transfection: At your bench, bring 2.0 μg total plasmid DNA up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room. In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at 37°C. Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet. 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. Remove the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet. Aspirate off the old antibiotic-containing medium in the wells. Do NOT scrape the bottom of the wells, as the cells need to remain adherent to the plate. Add 4mL of warm antibiotic-free growth medium to each well. 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. Transgene expression should be detectable after 18 hours.