User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/02/22: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav)
 
(2 intermediate revisions by one other user not shown)
Line 2: Line 2:
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
Line 13: Line 13:


'''DAPI/ luciferase staining'''<br>
'''DAPI/ luciferase staining'''<br>
Note: David, enter formulas, procedures here


* D-luciferin stock solution: ??? in 1xPBS
* D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x)
* Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O
* Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O


Line 30: Line 29:
* Did not detect D-luc signal. Perhaps D-luc imaging should start immediately after adding reagents. The D-luc signal has a short half-life.
* Did not detect D-luc signal. Perhaps D-luc imaging should start immediately after adding reagents. The D-luc signal has a short half-life.
* Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye.
* Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye.
* DAPI signal was barely visible




Line 45: Line 45:
* HEK293 Gal4-EED, 1:4
* HEK293 Gal4-EED, 1:4
* HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner)
* HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner)




Latest revision as of 22:29, 26 September 2017

Project name Main project page
Previous entry      Next entry

02/23/13

  • DAPI/ luciferase staining
  • New cell plates

DAPI/ luciferase staining

  • D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x)
  • Hoescht: made by Dr. Haynes; 5000x in sterile dH2O


  • Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well...

Used 3 wells:

  1. DAPI + D-luc
  2. DAPI only
  3. PBS
  • Let cells sit at room temperature for 10 minutes in the dark to develop signal

Results/ Conclusions:

  • 2 mL medium is susceptible to drying at outer wells; disrupts cell growth
  • Did not detect D-luc signal. Perhaps D-luc imaging should start immediately after adding reagents. The D-luc signal has a short half-life.
  • Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye.
  • DAPI signal was barely visible


Note: add photos here

New cell plates
Dr. Haynes; For more practice staining (Monday)

  • HEK293 luc #4, 6-well plate, ~1x105 cells/ well, 4 mL growth medium
  • HEK293 Gal4-EED, 6-well plate, ~1x105 cells/ well, 4 mL growth medium

Stock cultures (T75 flasks)

  • HEK293 luc #4, 1:5
  • HEK293 Gal4-EED, 1:4
  • HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner)




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:David_Dreher/Notebook/Chromatin_controlled_cell_pattern/2013/02/22&oldid=1012055"