Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</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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==Protocol== | ==Protocol== | ||
[http://openwetware.org/wiki/Haynes: | [http://openwetware.org/wiki/Haynes:FCMamalian_FluorGene flow cytometry of mammalian cells for measuring fluorescent gene expression]<br> | ||
[http://openwetware.org/wiki/Haynes:TRIzol_RNeasy RNA extraction / purification]<br><br> | |||
==Materials== | ==Materials== | ||
* | * PBS buffer | ||
* | * Trypsin / EDTA | ||
* Complete cell medium | |||
* FACS Buffer - 1% FBS in 1x PBS (stored at 4°C) | |||
* FACS tubes with strainer caps - 6 mL capacity (EMS 64750-25) | |||
* TRIzol / RNA purification miniprep & supplies | |||
* pipette tips, etc.<br><br> | |||
==Notes== | ==Notes== | ||
Overview of flow cytometry data: | |||
[[Image:20141223_flow_cytometry_overview.png | thumb]]<br><br> | |||
Negative control does not have significantly different fluorescence values than the samples (data not shown).<br><br> | |||
==Results/Conclusions== | ==Results/Conclusions== | ||
Since the control has the same fluorescence levels as the samples (and therefore the same gene expression levels of PcTF-mCherry), we will not be performing the RNA extraction / purification today. Instead we'll be repeating this experiment tomorrow on the second set of transfected cells.<br><br> | |||
*'''[[User:David Benjamin Nyer|David Benjamin Nyer]] | *'''[[User:David Benjamin Nyer|David Benjamin Nyer]] 17:27, 23 December 2014 (EST)''': | ||
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__NOTOC__ | __NOTOC__ | ||
Latest revision as of 00:36, 27 September 2017
 RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines
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SummaryToday we'll be harvesting the U2OS cells from the first transfection attempt. Fluorescence microscopy of the cells indicates there is little or no PcTF-RFP expression in the line, but we'll be doing a run through with flow cytometry and RNA purification regardless, as practice for future attempts. Goals
Protocolflow cytometry of mammalian cells for measuring fluorescent gene expression Materials
NotesOverview of flow cytometry data:  Negative control does not have significantly different fluorescence values than the samples (data not shown). Results/ConclusionsSince the control has the same fluorescence levels as the samples (and therefore the same gene expression levels of PcTF-mCherry), we will not be performing the RNA extraction / purification today. Instead we'll be repeating this experiment tomorrow on the second set of transfected cells.
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