06/10/10
- ✓ ChIP: 126-1 and U2OS plain samples (corrected protocol)
- ✓ PCR: test ChIP primers on U2OS plain quality control DNA
ChIP
> Do 4 IP's for each sample
> Samples:
- 126-1, ~1.7 mL/ IP
- U2OS plain (remainder from last attempt), ~1.2 mL/ IP
> Antibodies for IP
- mouse α-PolII 8WG16 (5 μg)
- rabbit α-myc ab9106 (5 μg)
- rabbit α-H3K27me3 07-449 (5 μg)
- mouse α-IgG sc-2025 (2 μg) + rabbit α-IgG sc-2027 (2 μg)
PCR
> Test ChIP PCR primers (expected amplicons in bp)
- INKARF A f1/r1 = 125
- INKARF B f1/r1 = 134
- INKARF C f1/r1 = 138
- INKARF D f1/r1 = 135
- INKARF E f1/r1 = 86
- INKARF F f1/r1 = 100
- INKARF G f1/r1 = 115
- INKARF H f1/r1 = 113
- INKARF I f1/r1 = 110
- ATF3 A f1/r1 = ?
- CCND2 A f1/r1 = ?
- GAPDH A f1/r1 = 117
- GAPDH B f1/r1 = 128
> Templates
- U2OS sonicated DNA quality control sample (from 5/11/2010)
- Flp-in T-REx genomic DNA
| Reagent |
Volume |
Master mix (x14)
|
15 μL/lane; 2% agarose
|
| DNA |
0.5 |
7.0
|
| 10μM primers |
1.0 |
---
|
| 2x GoTaq Green |
10.0 |
140.0
|
| dH2O |
8.5 |
119.0
|
| |
20.0 μL
|
|
--> Aliquot 19.0 μL of each master mix to appro. wells
--> Add 1.0 primers to each well
--> PCR (96-well)
- 95°C/ 3 min.
- [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
- 72°C/ 3 min.
- 4°C/ ∞
--> Success! All primer pairs work. DNA from sonicated chromatin can be amplified by all primer pairs.
|
Project name
Main project page
Previous entry Next entry
