10/19/10
- ✓ ChIP qPCR
- ✓ HEK Gal4EED: luciferase assays
ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 4.5 μL):
- KAH126-1 fx Input, pos (1:1)
- KAH126-1 fx myc IP, uk (1:1)
- KAH126-1 fx IgG, neg (1:1)
- KAH132-8 fx Input, pos (1:1)
- KAH132-8 fx myc IP, uk (1:1)
- KAH132-8 fx IgG, neg (1:1)
- KAH130-4 fx Input, pos (1:1)
- KAH130-4 fx myc IP, uk (1:1)
- KAH130-4 fx IgG, neg (1:1)
- 0 template, NTC (dH2O)
> Primers (30 rxns each):
--> Plate 1
- MMP12 A3
- MMP12 B2
--> Plate 2
- MMP12 C2
- MMP12 D3
--> Plate 3
- GAPDH A3
- GAPDH B2
--> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O
| Reagent |
1 rxn |
Primer mix (x31)
|
| ChIP DNA (1:1) |
4.5 |
---
|
| SYBR Green mix |
7.5 |
232.5
|
| 750 nM primers |
3.0 |
93.0
|
| dH2O |
--- |
---
|
| |
15.0
|
--> Aliquot 31.5 primer mix into 1st well of each triplicate
--> Add 13.5 (4.5 x 3) DNA to 31.5 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set
Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
- 95°C/ 5 min.
- [95°C/ 15 sec, 57°C/ 15 sec, 72°C/ 15 sec] x45
- Melt curve range 57°C -> 95°C/ 0.5°C per step
HEK Gal4-EED Luciferase assays
> Transfection looks good (~50%) for 5 and 7 day induced cells
> Very low transfection efficiency for 0 and 2 day induced cells
> Apirate off old medium. Resuspend cells in 1 mL new medium. Collect 500 uL cells into epi tubes (use 100 uL for luc assay (triplicates) and rest for cell counts)
> Re-plate remaining 500 uL in new plates. Bring medium vol up to 1.5 (non selective med.).
> Luciferase assay:
--> Plate 3 (5-day), samples 1-11*
--> Plate 4 (7-day), samples 1-11*
--> Gal4EED induction plate, samples 1-12 (3x 0 dox, 2 day, 5 day, 7 day)
> *Note: transfection of Gal4-ATF (KAH60) showed very low RFP signal
Conclusions:
- Expect to see low luc expression in dox-induced cells
- Looking for increased luc expression in Pc-ATF cells, not in controls
|
Project name
Main project page
Previous entry Next entry
