User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/30: Difference between revisions
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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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==6/30/13 | ==6/30/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* 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/luc cells | '''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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> 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 | > 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 | | 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 || " || " || " | ||
|} | |} | ||
(*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°C overnight<br> | > Grow cells at 37°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. | |||
Latest revision as of 22:53, 26 September 2017
Pc-TF Genomics | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6/30/13
Gal4-EED/luc cells +dox
Addition of serial dilutions of dox to cells
Lipo transfection
(*Ran out of plasmid DNA) > Add 570 μL Opti-MEM to each 20 μL DNA sample Results (7/01/13)
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