User:Karmella Haynes/Notebook/PcTF Genomics/2014/10/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 12: Line 12:
* Luc14 cells - Spinoculation for Jonah's samples (Biodesign)
* Luc14 cells - Spinoculation for Jonah's samples (Biodesign)
* K562 - Spinoculation (Haynes lab)
* K562 - Spinoculation (Haynes lab)
* Passages - SK-N-SH, 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.




Line 17: Line 42:
'''K562 - Spinoculation (Haynes lab)'''<br>
'''K562 - Spinoculation (Haynes lab)'''<br>
* Try spin technique on K562 cells using 15 mL conicals
* 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)
* Lipo LTX transfection (standard protocol)
* Take half of the transfected sample (2 mL), spin at 2000 rpm for 10 min, replate
* 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
* Final volumes for samples was 2mL. Did not bring up to 4 mL
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
|-
| reagent 1 || # μL
|-
| reagent 2 || #
|-
| reagent 3 || #
|-
| reagent 4 || #
|-
| dH<sub>2</sub>O || #
|-
| &nbsp; || # μL
|}
--> Reaction conditions




Revision as of 15:53, 18 October 2014

Pc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>


10/18/14

  • Luc14 cells - Spinoculation for Jonah's samples (Biodesign)
  • K562 - Spinoculation (Haynes lab)
  • Passages - SK-N-SH, 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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/PcTF_Genomics/2014/10/18&oldid=830010"