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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|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>}}
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==Entry title==
==02/23/13==
* Insert content here...
* DAPI/ luciferase staining
* New cell plates
 
----
 
'''DAPI/ luciferase staining'''<br>
 
* 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
 
 
* Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well...
Used 3 wells:
# DAPI + D-luc
# DAPI only
# 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'''<br>
Dr. Haynes; For more practice staining (Monday)
* HEK293 luc #4, 6-well plate, ~1x10<sup>5</sup> cells/ well, 4 mL growth medium
* HEK293 Gal4-EED, 6-well plate, ~1x10<sup>5</sup> 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)
 
 




Latest revision as of 22:29, 26 September 2017

Project name Main project page
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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)




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