User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30: Difference between revisions

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==mm/dd/yy==
==6/30/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
* Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
* PcTF transfection of SK-N-SH
 


----
----
'''Line item 1'''<br>
'''Gal4-EED/luc cells +dox'''<br>
> Samples
* Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
 
# 1.0 μg/mL dox
# 10<sup>-1</sup>
# 10<sup>-2</sup>
# 10<sup>-3</sup>
# 10<sup>-4</sup>
# 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...
 
{| {{table}}
|-
| &nbsp; || 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<sup>-1</sup> || 10<sup>-2</sup> || 10<sup>-3</sup> || 10<sup>-4</sup> || 0
|}
 
 
----
'''Lipo transfection'''<br>
> Samples are for RT-PCR
> Cells were 100% confluent<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; Note: no TetR in SK-N-SH, so expression will be constitutive
 


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
|-
|-
| reagent 1 || # μL
| <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 2 || #
| 1            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || 570 μL          || 2.5 μL          || 7.5 μL
|-
|-
| reagent 3 || #
| 2            || KAH126/MV2        || 2 μg      || 8.8 μL +11.2  || "            || "          || "
|-
|-
| reagent 4 || #
| 3            || <s>KAH126/MV2</s>*  || <s>2 μg</s>    || <s>8.8 μL +11.2</s>  || "            || "          || "
|-
|-
| dH<sub>2</sub>O || #
| 4            || mock        || none    || 0 μL + 20.0  || "            || "          || "
|-
|-
| &nbsp; || # μL
| 5            || mock        || none    || 0 μL + 20.0  || "            || "          || "
|-
| 6            || mock          || none      || 0 μL + 20.0          || "          || "          || "
|}
|}
(*Ran out of plasmid DNA)


--> 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 (7/01/13)
* Checked RFP expression on the Nikon scope
* Expression is good. Expand cells in 10 cm plates
* Do TRIzol prep after ~10 days.




Revision as of 20:12, 5 July 2013

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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.



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