|
07/04/13
- 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
Cell prep
- 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
Assay reagents
- Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)
Luc assay
- 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 6/18/13
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 |
|
Cells/ 100 μL
|
| 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
- Lower range worked very well for trail #1! Saw ranges in expression for 1.0 - 10-4 μg/mL doc after 4 days
- Repeat induction and test after 48 hours
- 1.0 μg/mL dox
- 10-1
- 10-2
- 10-3
- 10-4
- 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...
| |
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 |
10-1 |
10-2 |
10-3 |
10-4 |
0
|
|
Pc-TF Genomics
