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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)
- plate 1, well A - DNA
- plate 1, well B - mock
- plate 2, well A - DNA
- 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
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)
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Pc-TF Genomics
