User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/18: Difference between revisions
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* | * Luciferase activity assay | ||
---- | ---- | ||
''' | '''Luciferase activity assay - time point 1'''<br> | ||
> | |||
<u>Cell prep</u> | |||
* Harvested cells | |||
** Seeded new dox+ plate with 1/2 of cells | |||
** Pelleted other half, resuspended in 2 mL FACS buffer | |||
<u>Assay reagents</u> | |||
* Used the Biotium Steady-Luc Firefly HTS Assay Kit | |||
* Thawed reagents to room temp | |||
* 6 experimental samples x 3 repliates = '''18 assays (one time point)''' | |||
* Need 100 μL complete steady-luc buffer per assay = '''1800 μL complete steady-luc buffer''' | |||
* Dissolved dry D-luciferin to make 50x stock by adding 80 μL dH<sub>2</sub>O to the vial | |||
<u>Luc assay</u> | |||
* Filtered 500 μL cells through strainer caps (<font color="red">should use 600 μL next time</font>) | |||
* Transferred 100 μL strained cells to 3 wells in 96-well plate (black walls; clear, flat bottom) | |||
** Saved remainder of strained cells for flow cytometry/ counting | |||
* Made 2000 μL complete steady-luc buffer: Added 40 μL 50x D-luciferin to 2 mL buffer | |||
* QUICKLY Added 100 μL of complete steady-luc buffer to each sample (pipetted up/down once to mix) | |||
* Incubated at room temp/ 10 min. | |||
* Synergy H1 reader - Created new protocol: KAH_luciferase | |||
** Optimized specs: Luminescence, Endpoint, Full plate, Integration time 0:01.00 (MM:SS.ss), Filter set 1, Emission: full light, Optics: top, '''Gain 200''', Read Speed: normal, Delay: 100 msec, Extended Dynamic Range, Read Height: 1 mm | |||
<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 || 8077 || x5 = || 40,385 | ||
|- | |- | ||
| | | sample 2 || 9552 || x5 = || 47,760 | ||
|- | |- | ||
| | | sample 3 || 9460 || x5 = || 47,300 | ||
|- | |- | ||
| | | sample 4 || 3608 || x5 = || 18,040 | ||
|- | |- | ||
| | | sample 5 || 4723 || x5 = || 23,615 | ||
|- | |- | ||
| | | sample 6 || 5851 || x5 = || 29,255 | ||
|} | |} | ||
Revision as of 19:44, 18 June 2013
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06/18/13
Luciferase activity assay - time point 1
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