User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/06: Difference between revisions
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* | * Gal4-EED/luc dox time point (48 hours) | ||
* Gal4-EED/luc PcTF transfection - microscopy & flow cytometry | |||
* SK-N-SH +PcTF & mock - TRIzol prep #1 | |||
---- | ---- | ||
''' | '''Luciferase activity assay - time point: 2 days'''<br> | ||
> Samples | |||
<u>Cell prep</u> | |||
* Harvested cells (induced on 7/04/13) | |||
** Seeded new 2 new plates with non-induced cells from 7/04/13 | |||
** Pelleted all of the dox+ cells, resuspended in 2 mL FACS buffer | |||
<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/2013/06/18 6/18/13]) | |||
<u>Luc assay</u> | |||
* Filtered '''700 μL cells''' through strainer caps | |||
* Used opaque white Costar plate (from Rege lab) | |||
* Samples loaded in triplicate (by columns) | |||
* Included luc buffer + FACS-buffer "blank" sample (well D1) | |||
* Other steps same as [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/06/18 6/18/13] | |||
<u>Cell counts</u> | |||
* Wang lab's Accuri flow cytometer. | |||
* Set machine to read 20 uL of cells | |||
* Be sure to "clean" with water-run in between samples | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | ||
|-valign="top" | |-valign="top" | ||
| <u> | | <u>Sample ID</u> || <u>Gated count/ 20 μL</u> || || <u>Cells/ 100 μL</u> | ||
|- | |- | ||
| | | sample 1 || 37,629 || x5 = || 188,145 | ||
|- | |- | ||
| | | sample 2 || 34,580 || x5 = || 172,900 | ||
|- | |- | ||
| | | sample 3 || 40,096 || x5 = || 200,480 | ||
|- | |- | ||
| | | sample 4 || 46,599 || x5 = || 232,995 | ||
|- | |- | ||
| | | sample 5 || 46,491 || x5 = || 232,455 | ||
|- | |- | ||
| | | sample 6 || 46,254 || x5 = || 231,270 | ||
|} | |} | ||
--> | |||
---- | |||
'''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 | |||
Revision as of 04:33, 9 July 2013
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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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