User:Karmella Haynes/Notebook/PcTF Genomics/2014/10/08

Pc-TF Genomics

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10/08/14 "Spinoculation" transfection expriment "Spinoculation" transfection expriment Try centrifuge spin approach to increase transfection efficiency See notes from Use U2OS cells Samples KAH126/MV2, spin mock, spin KAH126/MV2 conventional mock conventional Spinoculation Set up Lipo-DNA and mock complexes for 6-well plates (standard Lipo LTX protocol): 2000 μg/ 20 μL DNA, 570 μL Opti-MEM, 2.5 μL PLUS, 7.5 μL Lipofectamine LTX Harvest U2OS cells from T-75 flask (~1.0E6 cells/ mL) Transfer ~5.0E5 cells into sterile 15 mL conicals. Fill up to final vol. of 3.4 mL with growth medium Add 0.6 mL Lipo-DNA or mock complexes. Swirl to mix. Spin at 3000 rpm (800 - 1000 xg) at room temp for 25 min. Note: centrifuge is IEC Centra-CL2 with rotor 215. Transfer most of supernatant into 6-well plate wells Flick to disrupt the pelleted cells. Use 1 mL of growth medium to transfer cells to wells. Conventional Remove 0.5 mL growth medium from each well Drop-wise, add Lipo-DNA or mock complexes to wells Grow all cells overnight at 37°C/5% CO2. Check for RFP expression tomorrow