User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30

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(Autocreate 2013/06/30 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
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==mm/dd/yy==
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==6/30/13/dd/yy==
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* Line item 1
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* Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
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* PcTF transfection of SK-N-SH
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* Gal4-EED ELISA set-up
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----
----
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'''Line item 1'''<br>
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'''Gal4-EED/luc cells IFC'''<br>
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> Samples
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* 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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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
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# 1.0 μg/mL dox
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|-valign="top"
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# 10<sup>-1</sup>
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| <u>Reagent</u> || <u>Volume</u>  
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# 10<sup>-2</sup>
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# 10<sup>-3</sup>
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# 10<sup>-4</sup>
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# 0
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----
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'''Lipo transfection'''<br>
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> Samples are for RT-PCR
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> Cells were 100% confluent<br>
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> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium<br>
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> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH126/MV2); ### ng/μL<br>; Note: no TetR in SK-N-SH, so expression will be constitutive
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 +
 
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{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
|-
|-
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| reagent 1 || # μL
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| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume + dH<sub>2</sub>O</u> || <u>Opti-MEM</u> || <u>PLUS</u> || <u>Lipo</u>  ||  
|-
|-
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| reagent 2 || #
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| 1            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || 570 μL          || 2.5 μL          || 7.5 μL
|-
|-
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| reagent 3 || #
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| 2            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || "            || "          || "
|-
|-
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| reagent 4 || #
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| 3            || KAH126/MV2        || 2 μg    || 8.8 μL +11.2  || "            || "          || "
|-
|-
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| dH<sub>2</sub>O || #
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| 4            || mock        || none    || 0 μL + 20.0  || "            || "          || "
|-
|-
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| &nbsp; || # μL
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| 5            || mock        || none    || 0 μL + 20.0  || "            || "          || "
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|-
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| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
|}
|}
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--> Reaction conditions
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> Add 570 μL Opti-MEM to each 20 μL DNA sample <br>
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> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp<br>
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> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp <br>
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> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)<br>
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> Grow cells at 37&deg;C overnight<br>
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Revision as of 17:01, 30 June 2013

Pc-TF Genomics Main project page
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6/30/13/dd/yy

  • 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 ELISA set-up



Gal4-EED/luc cells IFC

  • 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



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); ### 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 " " "

> 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




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