Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/27

Neon transfection of K562 cell line

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Overview Trying transfection of K562 again, this time using the Neon transfection system. Protocol Set up a 12 well plate with 1 mL of antibiotic-free medium in each well. Place in the incubator. Spin down 5 mL of K562 culture and wash with PBS. Check density using hemocytometer. Spin down again, resuspend in approximate volume of Resuspension Buffer R to get 1E7 cells/mL using Resuspension Buffer R. Check density again using hemocytometer. Follow the instructions on page 2 of the Neon Transfection System reference card to proceed. Samples: pMax GFP, 1000 v, 50 ms, 1 pulse pMax GFP, 1350 v, 10 ms, 4 pulses pMax GFP, 1450 v, 10 ms, 3 pulses KAH126-MV2, 1000 v, 50 ms, 1 pulse KAH126-MV2, 1350 v, 10 ms, 4 pulses KAH126-MV2, 1450 v, 10 ms, 3 pulses Treat samples in duplicate, for 12 samples in total. Notes Culture density: 240,000 cells/mL * 5 mL culture = 1.2E6 cells total To make 1E7 cells/mL concentration, spin down and resuspend in 120 µL Resuspension Buffer R. pMaxGFP concentration: 2000 ng/µL KAH126-MV2 concentration: 809 ng/µL