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 Pc-TF Genomics
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06/18/13
- Luciferase activity assay
Luciferase activity assay - time point 1
Cell prep
- Harvested cells
- Seeded new dox+ plate with 1/2 of cells
- Pelleted other half, resuspended in 2 mL FACS buffer
Assay reagents
- 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 dH2O to the vial
Luc assay
- Filtered 500 μL cells through strainer caps (should use 600 μL next time)
- 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
Cell counts
- Wang lab's Accuri flow cytometer.
- Set machine to read 20 uL of cells
- Be sure to "clean" with water-run in between samples
| Sample ID |
Gated count/ 20 μL |
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Cells/ 100 μL
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| sample 1 |
8077 |
x5 = |
40,385
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| sample 2 |
9552 |
x5 = |
47,760
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| sample 3 |
9460 |
x5 = |
47,300
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| sample 4 |
3608 |
x5 = |
18,040
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| sample 5 |
4723 |
x5 = |
23,615
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| sample 6 |
5851 |
x5 = |
29,255
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Pc-TF Genomics
