10/11/10
- ✓ ChIP qPCR: 126-1, 132-8 continued
- ✓ HepG2 culture: Split HepG2 for transfections; back-up stock 1:5
- ✓ HEK Gal4EED culture: induce 12-well plate with dox (set 1); back-up stock 1:5
ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 4.0 μL, 12 rxns each):
- KAH126-1 Input (#16), pos
- KAH126-1 αmyc IP (#18), unk
- KAH126-1 αIgG IP (#20), neg
- 0 template (dH2O)
- KAH132-8 Input (#21), pos
- KAH132-8 αmyc IP (#23), unk
- KAH132-8 αIgG IP (#25), neg
- 0 template (dH2O)
> Primers (24 rxns each):
- MMP12 C2
- TNF B
- TNF C3
- TNF C1
--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
Reagent |
1 rxn |
Primer mix (x25)
|
ChIP DNA (1:1) |
4.5 |
---
|
SYBR Green mix |
7.5 |
187.5
|
750 nM primers |
3.0 |
75.0
|
dH2O |
--- |
---
|
|
15.0
|
--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 13.5 DNA to primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in 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
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