User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/04: Difference between revisions
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== | ==07/04/13== | ||
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* | * Gal4-EED/luc time/dose - luc assay & cell counts | ||
* Happy July 4th! :) | |||
* Gal4-EED/luc Dox induction #2 | |||
---- | ---- | ||
''' | '''Luciferase activity assay - time point: 4 days'''<br> | ||
> Samples | |||
<u>Cell prep</u> | |||
* Harvested cells (induced on 6/30/13) | |||
** Seeded new 2 new plates with non-induced cells from 6/30/13 | |||
** 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 || 13,844 || x5 = || 69,220 | ||
|- | |- | ||
| | | sample 2 || 12,286 || x5 = || 61,430 | ||
|- | |||
| sample 3 || 15,784 || x5 = || 78,920 | |||
|- | |||
| sample 4 || 16,323 || x5 = || 81,615 | |||
|- | |||
| sample 5 || 16,545 || x5 = || 82,725 | |||
|- | |||
| sample 6 || 17,885 || x5 = || 89,425 | |||
|} | |||
---- | |||
'''Gal4-EED/luc Dox induction #2'''<br> | |||
* Lower range worked very well for trail #1! Saw ranges in expression for 1.0 - 10<sup>-4</sup> μg/mL doc after 4 days | |||
* Repeat induction and test after 48 hours | |||
# 1.0 μg/mL dox | |||
# 10<sup>-1</sup> | |||
# 10<sup>-2</sup> | |||
# 10<sup>-3</sup> | |||
# 10<sup>-4</sup> | |||
# 0 | |||
Addition of serial dilutions of dox to cells | |||
* Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary. | |||
* Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes... | |||
{| {{table}} | |||
|- | |- | ||
| | | || tube 1 || 2 || 3 || 4 || 5 || 6 | ||
|- | |- | ||
| | | 1 mg/mL dox (μL) || 8.0 || --- || --- || --- || --- || --- | ||
|- | |||
| mixture from prior tube (μL) || --- || 100 || 100 || 100 || 100 || --- | |||
|- | |- | ||
| | | +growth med. (μL) || 1000 || 900 || 900 || 900 || 900 || --- | ||
|- | |- | ||
| | | Final [dox] (μg/mL) in 4 mL med. || 1.0 || 10<sup>-1</sup> || 10<sup>-2</sup> || 10<sup>-3</sup> || 10<sup>-4</sup> || 0 | ||
|} | |} | ||
Revision as of 20:08, 6 July 2013
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07/04/13
Luciferase activity assay - time point: 4 days
Gal4-EED/luc Dox induction #2
Addition of serial dilutions of dox to cells
|