User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/06: 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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| colspan="2"| | | colspan="2"| | ||
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* Gal4-EED/luc dox time point (48 hours) | * Gal4-EED/luc dox time point (48 hours) | ||
* Gal4-EED/luc PcTF transfection - microscopy & flow cytometry | * Gal4-EED/luc PcTF transfection - microscopy & flow cytometry | ||
* SK-N-SH +PcTF & mock - TRIzol prep #1 | |||
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<u>Assay reagents</u> | <u>Assay reagents</u> | ||
* Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/ | * Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18 6/18/13]) | ||
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* Samples loaded in triplicate (by columns) | * Samples loaded in triplicate (by columns) | ||
* Included luc buffer + FACS-buffer "blank" sample (well D1) | * Included luc buffer + FACS-buffer "blank" sample (well D1) | ||
* Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/ | * Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18 6/18/13] | ||
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| <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || || <u>Cells/ 100 μL</u> | | <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || || <u>Cells/ 100 μL</u> | ||
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| sample 1 || 37,629 || x5 = || | | sample 1 || 37,629 || x5 = || 188,145 | ||
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| sample 2 || | | sample 2 || 34,580 || x5 = || 172,900 | ||
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| sample 3 || | | sample 3 || 40,096 || x5 = || 200,480 | ||
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| sample 4 || | | sample 4 || 46,599 || x5 = || 232,995 | ||
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| sample 5 || | | sample 5 || 46,491 || x5 = || 232,455 | ||
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| sample 6 || | | sample 6 || 46,254 || x5 = || 231,270 | ||
|} | |} | ||
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'''SK-N-SH TRIzol prep'''<br> | |||
# +PcTF, plate 1 | |||
# mock, plate 4 | |||
(Cells may need longer to express genes. Previous expt. was done after ~10 days. Will do this prep just in case cell culture declines) | |||
* Used other two plates to passage cells ~1:5 (will prep after returning from SB6.0) | |||
* Cells were 100% confluent | |||
* Discarded growth medium | |||
* Added 2 mL TRIzol directly to plates | |||
* Incubated at r.t. for ~5 min. | |||
* Collected lysed cells from plate with gentle scraping (pipette tip) | |||
* Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C | |||
** 4x PcTF+ samples | |||
** 4x mock samples | |||
Latest revision as of 22:58, 26 September 2017
Pc-TF Genomics | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||
07/06/13
Luciferase activity assay - time point: 2 days Cell prep
SK-N-SH TRIzol prep
(Cells may need longer to express genes. Previous expt. was done after ~10 days. Will do this prep just in case cell culture declines)
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