10/03/10
- ✓ ChIP qPCR: experimental sample set 126-1
- ✓ Western: ChIP/ Co-IP for 126-1 (DsRed and H3K27me3)
- ✓ Culture: split HepG2's 1:10
qPCR
> Set up each reaction in triplicate
> Templates (use 4.5 μL):
- KAH126-1 Input, pos (1:25)
- KAH126-1 αmyc IP, exp (1:25)
- KAH126-1 αIgG IP, neg (1:25)
- 0 template (dH2O)
> Primers (12 rxns each):
- INKARF D
- INKARF E
- INKARF F
- INKARF G
- MMP12 A
- MMP12 B
- MMP12 C
- GAPDH B
--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
Reagent |
1 rxn |
x3 |
Primer mix (x13)
|
ChIP DNA (1:50) |
4.5 |
12.5 |
---
|
SYBR Green mix |
7.5 |
22.5 |
97.5
|
750 nM primers |
3.0 |
9.0 |
39.0
|
dH2O |
--- |
--- |
---
|
|
15.0
|
--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 12.5 1:25 template DNA to primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in 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/ 3°C per step
> Conclusions:
- C(t) still not very far from background. Try again using 2.0 μL 1:1 template
Western blot: ChIP/ Co-IP
> IP samples from 10/01/10; 30 μL each, ready to load
> Prep input and sup samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.
Gels 1 & 2
1. PageRuler Plus (10 μL)
2. KAH126-1 input
3. KAH126-1 α-myc sup
4. KAH126-1 α-myc IP
5. KAH126-1 α-IgG sup (neg ctrl)
6. KAH126-1 α-IgG IP (neg ctrl)
7. α-myc beads (neg ctrl)
8. PageRuler
9. dye
10. dye
Note: Use IP samples A or B for gel 1/2 (IP matched with appropriate sup)
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10/03/10 Ponceau S stain Ponceau S stained filters
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> Block: 5% milk/PBST, 4°C/overnight
10/04/10
> Primary staining: 5% BSA/PBST, 4°C/o.n.
- rabbit α-DsRed 632496, 1:1000, 5 mL
- rabbit α-H3K27me3 07-449, 1:500, 5 mL
10/05/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
- donkey α-rabbit-HRP, 1:5000; 5 mL
- donkey α-rabbit-HRP, mouse α-GAPDH-HRP 1:5000; 5 mL
> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
- KAH126-1: 43 kD
- H3K27me3: 15 - 17 kD
Western blot 10/05/10
Conclusions:
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