12/08/10
- ✓ ChIP qPCR: re-do FTRx fx INKARF F1
ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 2.0 μL, duplicate in., K28, & myc):
- FTRx fx input, pos
- FTRx fx H3K27me3 IP, uk
- FTRx fx myc, neg
- FTRx fx input, pos
- FTRx fx H3K27me3 IP, uk
- FTRx fx myc, neg
- 0 template
> Primers (21 rxns pre primer pair):
--> Plate 2
- INKARF F1
--> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O
Reagent |
1 rxn |
Primer mix (x23)
|
ChIP DNA |
2.0 |
---
|
SYBR Green mix |
7.5 |
172.5
|
750 nM primers |
3.0 |
69.0
|
dH2O |
2.5 |
57.5
|
|
15.0
|
--> Aliquot 39.0 primer mix into 1st well of each triplicate
--> Add 6.0 (2.0 x 3) DNA to 39.0 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
|