04/21/11
- ✓ Luc assay: HEK-luc rep +Pc-TF transfections (plate 2A)
- ✓ ChIP qPCR: Plates ch4
ChIP qPCR
> First check for enrichment of H3K27me3 at putative target genes
> Set up each reaction 4x
>Plate ch4
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):
- FTRx (29) input, pos
- FTRx (38) H3K27me3 IP, uk
- FTRx (39) IgG IP, neg
--> 750 nM primers (12 rxns per primer pair):
- NPPA A1
- NPPA B2
- NPPA C2
- NPPA D1
- EOMES A1
- EOMES B2
- EOMES C3
- EOMES D3
Reagent |
1 rxn |
Primer mix (x13)
|
ChIP DNA |
2.0 |
---
|
SYBR Green mix |
7.5 |
97.5
|
750 nM primers |
3.0 |
39.0
|
dH2O |
2.5 |
32.5
|
|
15.0
|
--> Aliquot 52.0 primer mix into 1st well of each 4x set
--> Add 8.0 (2.0 x4) DNA to 52.0 primer mix
--> Aliquot 15.0 rxn mix to other 3 wells in each 4x set
Bio-Rad CFX96 qPCR (Kirschner lab)
--> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
- 95°C/ 5 min.
- [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
- Melt curve range 58°C -> 95°C/ 0.5°C per step
|