04/13/11
- ✓ qRT-PCR: Pc-TF -/+dox plates 19, 20
- ✓ PCR: ChIP primer optimization (new primers)
qRT-PCR
> Plate 19
--> 750 nM Primers (& cDNA dilution)
- mCh1 (1:1000)
- GAPDH 21A (1:1000)
- ACTC1 35A (1:10)
- ATOH1 46D "
- CASP10 41A "
- CDKN2A 7C "
- EOMES 42A "
- GRHL2 37C "
> Templates, use 2 μL
- KAH154-2, 4/08/11 A
- KAH154-2 +dox, 4/08/11 A
- KAH154-2, 4/08/11 B
- KAH154-2 +dox, 4/08/11 B
> Plate 20
--> 750 nM Primers (& cDNA dilution)
- MMP12 31B (1:10)
- NPPA 40A "
- OTX2 43D "
- SST 45C "
- THPO 33A "
- TNF 34A "
> Templates, use 2 μL
- KAH154-2, 4/08/11 A
- KAH154-2 +dox, 4/08/11 A
- KAH154-2, 4/08/11 B
- KAH154-2 +dox, 4/08/11 B
Reagent |
1 rxn |
Primer mixes 1-6 (x13)
|
diluted cDNA |
2.0 |
---
|
SYBR Green mix |
7.5 |
97.5
|
750 nM primers |
3.0 |
39.0
|
dH2O |
2.5 |
32.5
|
|
15.0
|
--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) 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, 58°C/ 15 sec, 72°C/ 15 sec] x45
- Melt curve range 58°C -> 95°C/ 0.5°C per step
PCR: ChIP primer optimization
> Test new ChIP primers on old DNA sample (from sonicated CH2O x-linked chromatin)
> Primers (10 μM mix)
- 1-12. ATOH1 A1-3, B1-3, C1-3, D1-3
- 13-24. CASP10 A1-3, B1-3, C1-3, D1-3
- 25-36. EOMES A1-3, B1-3, C1-3, D1-3
- 37-48. GRHL2 A1-3, B1-3, C1-3, D1-3
- 49-60. NPPA A1-3, B1-3, C1-3, D1-3
- 61-72. OTX2 A1-3, B1-3, C1-3, D1-3
- 73-84. ACTC1 A1-3, B1-3, C1-3, D1-3
> Template (use 2 μL)
- 130-4 Input, FX (#6), 9/15/10)
Reagent |
1 rxn |
Template mix (x86)
|
ChIP input DNA |
2.0 |
172.0
|
2x GoTaq Green mix |
10.0 |
860.0
|
10 μM primers |
1.0 |
---
|
dH2O |
7.0 |
602.0
|
|
20.0
|
- Note: use 2.0 mL tube for master mix
Bio-Rad 96-well
- 95°C/ 3 min.
- [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x32
- 72°C/ 3 min.
- 4°C/ ∞
> Conclusions: Test the following via qPCR (template vs. no template)
- ATOH1 A1, B3, C2, D1
- CASP10 A1, B1, C3, D2
- EOMES A1, B2, C3, D3
- GRHL2 A1, B1, C1, D1
- NPPA A1, B2, C2, D1
- OTX2 A1, B2, C1, D1
- ACTC1 A1, B1, C2, D1
|