04/26/11
- ✓ ChIP qPCR: plates ch6, ch7, ch8, ch9
ChIP qPCR
> NPPA and EOMES showed H3K27me3 enrichment; check for Pc-TF enrichment
> Set up each reaction 4x
>Templates
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):
- 132-8 (21) input, pos
- 132-8 (36) myc IP, uk
- 132-8 (37) IgG IP, neg
- 126-1 (16) input, pos
- 126-1 (32) myc IP, uk
- 126-1 (33) IgG IP, neg
- 128-8.3 (40) input, pos
- 128-8.3 (41) myc IP, uk
- 128-8.3 (42) IgG IP, neg
- 129-4 (43) input, pos
- 129-4 (44) myc IP, uk
- 129-4 (45) IgG IP, neg
> Plate ch6 primers
--> 750 nM primers (48 rxns per primer pair):
- EOMES A1
- EOMES B2
> Plate ch7 primers
--> 750 nM primers (48 rxns per primer pair):
- EOMES C3
- EOMES D3
> Plate ch8 primers
--> 750 nM primers (48 rxns per primer pair):
- NPPA B2
- NPPA C2
> Plate ch9 primers
--> 750 nM primers (48 rxns per primer pair):
- NPPA A1
- NPPA D1
| Reagent |
1 rxn |
Primer mix (x49)
|
| ChIP DNA |
2.0 |
---
|
| SYBR Green mix |
7.5 |
367.5
|
| 750 nM primers |
3.0 |
147.0
|
| dH2O |
2.5 |
122.5
|
| |
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
|
Project name
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