User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/12: 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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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Gal4-EED/luc cells IFC - stain with anti-luc overnight | * Gal4-EED/luc cells IFC - stain with anti-luc overnight | ||
* PcTF transfection of U2OS | |||
---- | ---- | ||
'''Gal4-EED/luc cells IFC'''<br> | '''Gal4-EED/luc cells IFC'''<br> | ||
* Samples: first | * Samples: first row of wells in the 24-well plate. | ||
* Keep all others under PBS | * Keep all others under PBS | ||
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# 0.5 | # 0.5 | ||
# 1.0 | # 1.0 | ||
# 2.0 | # 2.0 (induced on 6/02/13, 10 days) | ||
* Use 1:200 dilution of rabbit (poly) anti-luc (in 15 μL Gentex horse serum blocking solution per well) | |||
* Incubate under parafilm "covers" overnight at 4°C in box with wet paper towels | |||
---- | |||
'''Lipo transfection'''<br> | |||
> 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 promoter (KAH160/MV1); 346.6 ng/μL<br> | |||
> Samples are for RT-PCR | |||
{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table --> | |||
|- | |||
| <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> || | |||
|- | |||
| 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 || KAH160/MV1 || 2 μg || 5.8 μL +14.2 || " || " || " | |||
|- | |||
| 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 <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°C overnight<br> | |||
Latest revision as of 22:45, 26 September 2017
Pc-TF Genomics | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||
06/12/13
Gal4-EED/luc cells IFC
Lipo transfection
> Add 570 μL Opti-MEM to each 20 μL DNA sample
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