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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</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>}}
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|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}}}}]]}}
 
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==6/30/13/dd/yy==
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==6/30/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
* Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
 
* Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
 
* PcTF transfection of SK-N-SH
 
* PcTF transfection of SK-N-SH
* Gal4-EED ELISA set-up
 
  
  
 
----
 
----
'''Gal4-EED/luc cells IFC'''<br>
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'''Gal4-EED/luc cells +dox'''<br>
 
* Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
 
* Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
  
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| 1 mg/mL dox (μL) || 8.0 || --- || --- || --- || --- || ---
 
| 1 mg/mL dox (μL) || 8.0 || --- || --- || --- || --- || ---
| -  
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|-  
 
| mixture from prior tube (μL) || --- || 100 || 100 || 100 || 100 || ---
 
| mixture from prior tube (μL) || --- || 100 || 100 || 100 || 100 || ---
 
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> Cells were 100% confluent<br>
 
> Cells were 100% confluent<br>
 
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
 
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH126/MV2); 226 ng/μL<br>; Note: no TetR in SK-N-SH, so expression will be constitutive
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> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH126/MV2); 226 ng/μL; Note: no TetR in SK-N-SH, so expression will be constitutive
  
  
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| 2            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || "            || "          || "
 
| 2            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || "            || "          || "
 
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| 3            || KAH126/MV2         || 2 μg    || 8.8 μL +11.2  || "            || "          || "
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| 3            || <s>KAH126/MV2</s>*  || <s>2 μg</s>     || <s>8.8 μL +11.2</s> || "            || "          || "
 
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| 4            || mock        || none    || 0 μL + 20.0  || "            || "          || "
 
| 4            || mock        || none    || 0 μL + 20.0  || "            || "          || "
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| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
 
| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
 
|}
 
|}
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(*Ran out of plasmid DNA)
  
 
> Add 570 μL Opti-MEM to each 20 μL DNA sample <br>
 
> Add 570 μL Opti-MEM to each 20 μL DNA sample <br>
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> Grow cells at 37&deg;C overnight<br>
 
> Grow cells at 37&deg;C overnight<br>
  
 
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Results (7/01/13)
 +
* Checked RFP expression on the Nikon scope
 +
* Expression is good. Expand cells in 10 cm plates
 +
* Do TRIzol prep after ~10 days.
  
  

Latest revision as of 21:53, 26 September 2017

Owwnotebook icon.pngPc-TF Genomics Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


6/30/13

  • Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
  • PcTF transfection of SK-N-SH



Gal4-EED/luc cells +dox

  • Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
  1. 1.0 μg/mL dox
  2. 10-1
  3. 10-2
  4. 10-3
  5. 10-4
  6. 0

Addition of serial dilutions of dox to cells

  • Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary.
  • Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...
  tube 1 2 3 4 5 6
1 mg/mL dox (μL) 8.0 --- --- --- --- ---
mixture from prior tube (μL) --- 100 100 100 100 ---
+growth med. (μL) 1000 900 900 900 900 ---
Final [dox] (μg/mL) in 4 mL med. 1.0 10-1 10-2 10-3 10-4 0



Lipo transfection
> Samples are for RT-PCR > Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH126/MV2); 226 ng/μL; Note: no TetR in SK-N-SH, so expression will be constitutive


Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo
1 KAH126/MV2 2 μg 8.8 μL +11.2 570 μL 2.5 μL 7.5 μL
2 KAH126/MV2 2 μg 8.8 μL +11.2 " " "
3 KAH126/MV2* 2 μg 8.8 μL +11.2 " " "
4 mock none 0 μL + 20.0 " " "
5 mock none 0 μL + 20.0 " " "
6 mock none 0 μL + 20.0 " " "

(*Ran out of plasmid DNA)

> Add 570 μL Opti-MEM to each 20 μL DNA sample
> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)
> Grow cells at 37°C overnight

Results (7/01/13)

  • Checked RFP expression on the Nikon scope
  • Expression is good. Expand cells in 10 cm plates
  • Do TRIzol prep after ~10 days.