User:Karmella Haynes/Notebook/PcTF Genomics/2014/10/18

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngPc-TF Genomics Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


10/18/14

  • Luc14 cells - Spinoculation for Jonah's samples (Biodesign)
  • K562 - Spinoculation (Haynes lab)
  • Passages - SK-N-SH, U2OS, Gal4EED/Luc +dox



Luc14 cells - Spinoculation for Jonah's samples (Biodesign)

Transfections set up by Jonah on Friday (yesterday). Note: cells were in p/s+ medium (complete)

  1. plate 1, well A - DNA
  2. plate 1, well B - mock
  3. plate 2, well A - DNA
  4. plate 2, well B - DNA
  • Took plates over to Biodesign (LaBaer lab)
  • Spun plate 2 at 2250 rpm for 25 min.
  • Results...
    • 1A - viable cells, RFP expression is visible, but dim (800 ms exposure); note, only ~1100 μg DNA transfected because of low miniprep yield
    • 1B - viable cells, no RFP
    • 2A - very sick cells, ~10% survival; RFP expression is visible, but dim (800 ms exposure) in healthy cells
    • 2B - viable cells, no RFP

Conclusions:

  • Cells could have been vulnerable b/c of pen-strep.
  • I have tried the spin technique before with Lipo, but in p/s-free medium.
  • Should try again, but in p/s-free medium.



K562 - Spinoculation (Haynes lab)

  • Try spin technique on K562 cells using 15 mL conicals
  • Spin and resuspend cells to be transfected in p/s-free medium
  • Lipo LTX transfection (standard protocol)
  • Take half of the transfected sample (2 mL), spin at 2000 rpm for 10 min, replate
  • Final volumes for samples was 2mL. Did not bring up to 4 mL



Passages - SK-N-SH, U2OS, Gal4EED/Luc +dox

SK-N-SH

  • T-75: 1:5
  • 6-well plate: 0.5 mL per well (will switch to p/s-free before spinoculation transfection)

U2OS

  • T-75: 1:5
  • T-75: 1:10

Gal4EED/Luc

  • T-75: 2x 1:5 + 1μg/mL dox
  • 2x 6-well: 0.5 mL per well (will switch to p/s-free before spinoculation transfection)