Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/19

Setup for K562 transfection optimization

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Overview K562 cells are in PS-α right now. Once they're ready for expansion into PS-β, I'll be setting aside a fraction to grow in antibiotic-free medium for a transfection optimization experiment. Here's the general layout of the transfection procedure I'll be performing. Procedure 1. Prepare master mixes of Opti-MEM, DNA, & PLUS Reagent. Sample ID Opti-MEM (µL) DNA (µg) PLUS (µL) 1 50 4 5 2 50 1 5 3 50 0.4 5 4 50 6 5 5 50 1.5 5 6 50 0.6 5 7 50 8 5 8 50 2 5 9 50 0.8 5 10 50 10 5 11 50 2.5 5 12 50 1.0 5 2. Prepare master mixes of Opti-MEM and LTX Reagent. Sample ID Opti-MEM (µL) LTX (µL) 1 50 2 2 50 2 3 50 2 4 50 3 5 50 3 6 50 3 7 50 4 8 50 4 9 50 4 10 50 5 11 50 5 12 50 5 3. Incubate at room temperature for 5 minutes, then combine the two master mixes for each sample (100+µL total for each sample). 4. Take cells out of the incubator and add 50 µL of mixed DNA + LTX into each well (duplicates for each sample).