Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/09/01

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Overview

I'm going to be performing a series of transfections with the following parameters:

Cell type:

  • U-2 OS
  • SK-N-SH
  • K562

Time points:

  • post-24 hr
  • post-48 hr
  • post-72 hr

Replicates:

  • 2 biological replicates
  • 1 negative control

Vector used: KAH126-MV2

Other notes:

  • Take daily photographs of cells using fluorescence microscopy (phase/mCherry)
  • When harvesting cells, collect all supernatant from rinsing/washing steps and spin down with cells to harvest any non-adherent cells
  • Before treating samples with TRIzol, run on flow cytometry and measure the % efficiency via red fluorescence compared to the negative control

U-2 OS Transfection

Earlier in the week, I set up a 6-well plate with U-2 OS cells, and today I transfected the plate with KAH126-MV2 using Lipofectamine LTX. Wells 1, 2, 4, and 5 were treated with 4 µg of plasmid, and wells 3 and 6 were negative controls (no plasmid). After transfection, the plate was centrifuged at 128 g for 30 minutes at room temperature before being transferred to the CO2 incubator.