Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/08/11

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Overview

Performing two transfections today:
✓SK-N-SH in 6-well plates, using lipofectamine and KAH126-MV2
✓SK-N-SH in 24-well plates, using neon transfection and pMax-GFP for a test transfection

KAH126-MV2 Lipofectamine Transfection

SK-N-SH was grown until 90% confluent. Fresh EMEM w/ 10% FBS and no pen/strep was added. 4 wells were treated with 2 µg KAH126-MV2 plasmid and 2 wells were treated with no plasmid control. The plate was then centrifuged at 128g at 24°C for 30 minutes and then placed in the incubator. Tomorrow: replace medium with fresh antibiotic-free medium.

pMax-GFP Neon Transfection

SK-N-SH was harvested from a 75% confluent T-75 flask and resuspended in 1 mL PBS. Cell density measured at 1.1E6 cells/mL via flow cytometry. Cells were then spun down and resuspended in 55 µL of Neon transfection resuspension buffer R, for a final cell density of 2.0E7 cells/mL. Six samples were then made (three cell densities, in duplicate):

  • 2.0E7 cells/mL
  • 1.0E7 cells/mL
  • 5.0E6 cells/mL

Each sample was then given 2 µg of pMax-GFP plasmid and treated using the Neon transfection using one of the two following protocols:

  1. 900V, 50ms, 1 pulse
  2. 950V, 40ms, 1 pulse

These protocols are labeled "SKNMCP1" and "SKNMCP2" in the Neon transfection control box. Tomorrow: check for green fluorescence using the Nikon-TE fluorescence microscope.