12/21/10
- ✓ ChIP qPCR: continued from 12/20/10
- ✓ Colony growth assay: take bright field photos for 126-1 (~day 5); ~6.25x10^3 cells/ well showed good isolated colonies; will quantify cells per colony
- ✓ ChIP: wash & elute 128-8, 129-4 (store at -20°C until after vacation)
ChIP qPCR
> Set up each reaction 4x
> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):
- 126-1 (16) input, pos
- 126-1 (32) myc IP, uk
- 126-1 (33) IgG IP, neg
- 130-4 (06) input, pos
- 130-4 (34) myc IP, uk
- 130-4 (35) IgG IP, neg
- 132-8 (21) input, pos
- 132-8 (36) myc IP, uk
- 132-8 (37) IgG IP, neg
- FTRx (29) input, pos
- FTRx (38) H3K27me3 IP, uk
- FTRx (39) IgG IP, neg
> Primers (48 rxns per primer pair):
--> Plate 3
- GAPDH A3
- GAPDH B2
--> Plate 4
- MMP12 A3
- MMP12 B2
--> Plate 5
- MMP12 C2
- MMP12 D3
--> Plate 6
- TNF A2
- TNF B2
--> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O
Reagent |
1 rxn |
Primer mix (x50)
|
ChIP DNA |
2.0 |
---
|
SYBR Green mix |
7.5 |
375.0
|
750 nM primers |
3.0 |
150.0
|
dH2O |
2.5 |
125
|
|
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)
--> 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
ChIP: wash & elute IP's
> Last ChIP qPCR showed higher than desired mock IP background. Try to reduce using more washes.
> Washes: Follow Casey's (modified) protocol for all samples; use 2x washes instead of one (see 12/01/10)
> Elution & DNA purification: Follow Qingqing's protocol (see 12/01/10). Stop after TE elution. Keep supernatant at -20°C until after vacation.
> Samples (all form. x-linked chromatin)
1/2. 128-8: + 30 μL αmyc-beads (3400); + 30 μL mouse IgG-beads (3420)
3/4. 129-4: "
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