User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/05: Difference between revisions

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==mm/dd/yy==
==07/05/13==
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* Line item 1
* HEK293 Gal4-EED transfection - for PcTF expression and flow cytometry
 


----
----
'''Line item 1'''<br>
'''Lipo transfection'''<br>
> Samples
> Samples are for flow cytometry
> Seed new 6-well plates w/ ~5x10<sup>5</sup> cells/well in 3.4 mL plain medium (don't have any p.s.-free)<br>
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH160/MV1); 347 ng/μL; Note: this cell line has TetR, so constitutive CMV is needed.


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
 
|-valign="top"
{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
| <u>Reagent</u> || <u>Volume</u>
|-
| reagent 1 || # μL
|-
|-
| reagent 2 || #
| <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>  ||  
|-
|-
| reagent 3 || #
| 1            || KAH160/MV1        || 2 μg      || 5.8 μL +14.2  || 570 μL          || 2.5 μL          || 7.5 μL
|-
|-
| reagent 4 || #
| 2            || KAH160/MV1        || 2 μg      || 5.8 μL +14.2  || "            || "          || "
|-
|-
| dH<sub>2</sub>O || #
| 3            || mock  || none    ||  0 μL + 20.0 || "            || "          || "
|-
|-
| &nbsp; || # μL
| 4            || none        || none    || ---  || ---            || ---          || ---
|}
|}


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


Results (7/06/13)
* Success! Very high levels of RFP expression in wells 1 & 2 (microscopy), zero signal in mock and untreated (3 & 4)
* Harvest cells for flow cytometry




Revision as of 15:00, 6 July 2013

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07/05/13

  • HEK293 Gal4-EED transfection - for PcTF expression and flow cytometry


Lipo transfection
> Samples are for flow cytometry > Seed new 6-well plates w/ ~5x105 cells/well in 3.4 mL plain medium (don't have any p.s.-free)
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH160/MV1); 347 ng/μL; Note: this cell line has TetR, so constitutive CMV is needed.


Wells Plasmid DNA Volume + dH2O Opti-MEM PLUS Lipo
1 KAH160/MV1 2 μg 5.8 μL +14.2 570 μL 2.5 μL 7.5 μL
2 KAH160/MV1 2 μg 5.8 μL +14.2 " " "
3 mock none 0 μL + 20.0 " " "
4 none none --- --- --- ---

> 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 (7/06/13)

  • Success! Very high levels of RFP expression in wells 1 & 2 (microscopy), zero signal in mock and untreated (3 & 4)
  • Harvest cells for flow cytometry



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