10/18/10
- ✓ HepG2: split cells 1:5 in 75 cm flasks
- HEK Gal4EED: luciferase assay on hold (waiting for assay buffer)
- ✓ HEK Gal4EED: Use one Gal4EED induction plate to set up 12-well transfection plates; split 2 wells over 12 wells; split remaining well over three wells in original plate
- ✓ ChIP qPCR: KAH 126-1, 132-8, 130-4 IPs; INKARF D1, E2, F1, G3 primers
- ✓ Transfection: Fugene transfection for Gal4EED-induced HEK cells
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
- INKARF D1
- INKARF E2
--> Plate 2
- INKARF F1
- INKARF G3
--> 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
Transfections
> HEK293 Gal4-EED 23;4;9 cells, dox induced
> Use Fugene, 12-well format, 1.5 mL medium per well
> 4 plates total
- 0 dox (no Gal4-EED induction)
- 48 hr. dox (2 days)
- 120 hr. dox (5 days)
- 168 hr. dox (7 days)
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1. |
KAH126/MV2 |
500 ng |
1.0 μL |
1.5 μL |
48.5 μL
|
| 2. |
KAH154/MV2 |
" |
2.0 μL |
" |
"
|
| 3. |
KAH128/MV2 |
" |
1.0 μL |
" |
"
|
| 4. |
KAH157/MV2 |
" |
2.0 μL |
" |
"
|
| 5. |
KAH129/MV2 |
" |
1.0 μL |
" |
"
|
| 6. |
KAH156/MV2 |
" |
2.0 μL |
" |
"
|
| 7. |
KAH130/MV2 |
" |
1.5 μL |
" |
"
|
| 8. |
KAH158/MV2 |
" |
1.5 μL |
" |
"
|
| 9. |
KAH132/MV2 |
" |
1.0 μL |
" |
"
|
| 10. |
KAH60/pcVN |
" |
1.0 μL |
" |
"
|
| 11. |
no DNA |
--- |
--- |
" |
"
|
> Master mix (4x, 1 per plate): 6 μL Fugene + 194 μL Opti-MEM --> R.T./ 5 min.
> Add 4x vol. DNA --> R.T./ 20 min.
> Remove 0.5 mL medium from each well (decrease volume to improve transfection efficiency)
> Add 50 μL DNA/Fugene mix to each well (1.5 ml medium/ well); Grow cells at 37°C
> Refresh ab-free medium after 5 hours
> Check RFP/ luciferase after 18-24 hours
|
Project name
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