12/06/10
- ✓ ChIP qPCR 1: H3K27me3 IP set; optimize template load
- ✓ ChIP qPCR 2: H3K27me3 IP set; INKARF primers
- ✓ In vitro H3 tail binding assay: order H3K9me3 peptide and streptavidin beads
- ✓ Cell culture: thaw KAH126-1, 128-8.3, 129-4, 130-4 for histone peptide binding assays
- ✓ HepG2 TREx: change medium to DMEM plain (blast slowing cell growth?)
ChIP qPCR 1
> Set up each reaction in triplicate
> Templates (use 1, 2, 4.5 μL):
- FTRx dx input, pos (9 wells)
- FTRx dx H3K27me3 IP, uk (9)
- FTRx dx myc, neg (9)
- FTRx fx input, pos (9) *
- FTRx fx H3K27me3 IP, uk (9) *
- FTRx fx myc, neg (9)
- 0 template (3)
Note: Accidentally switched fx input and K27
> Primers (57 rxns total):
--> Plate 1
- GAPDH B2
--> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O
| Reagent |
1 rxn |
Primer mix (x60)
|
| ChIP DNA |
up to 4.5 |
---
|
| SYBR Green mix |
7.5 |
450.0
|
| 750 nM primers |
3.0 |
180.0
|
| dH2O |
--- |
---
|
| |
15.0
|
--> Aliquot 31.5 primer mix into 1st well of each triplicate
--> Add 13.5 (4.5 x 3) DNA+dH2O to 31.5 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
> Conclusion: GAPDH all volumes give C(t) = 27 for all input samples. 2 μL gives C(t) of about 33 - 35 for others. Melt curve peaks = 88.0 for all samples. Peak heights below threshold for K27me3 and myc IP as expected. Use 2 μL for future experimental samples.
ChIP qPCR 2
> Set up each reaction in triplicate
> Templates (use 2.0 μL):
- FTRx dx input, pos
- FTRx dx H3K27me3 IP, uk
- FTRx dx 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 D1
- INKARF E2
- INKARF F1
- INKARF G3
--> 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
|
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