12/14/11
- ✓ Illumina ChIp-seq prep: Pc-TF ChIP samples 1-6 (Day two)
Illumina ChIp-seq prep
> See 12/13/11 for list of samples (1-6)
Size Select the Library
> Run all samples (+ loading buffer) in a 2% TAE gel
> Use 100 bp ladder to excise area from 150 - 250
> Purify DNA with a Zymoclean Gel DNA recovery kit
> Elution: 2x 10 μL, add 16 μL st.dH2O to eluate (36 μL total)
Enrich the Adapter-Modified DNA Fragments by PCR
Reagent |
Volume |
Master mix (6x)
|
DNA |
36.0 μL |
---
|
5x Phusion buffer |
10.0 |
60.0
|
10 mM dNTP mix |
1.5 |
9.0
|
PCR primer 1.1 |
1.0 |
6.0
|
PCR primer 2.1 |
1.0 |
6.0
|
Phusion polymerase |
0.5 |
3.0
|
|
50.0 μL
|
--> Xiao's PCR machine, block A (heat lid 100°C):
- 98°C, 30 sec
- 18x [98°C, 10 sec/ 65°C, 30 sec/ 72°C, 30 sec]
- 72°C, 5 min
- 4°C, ∞
--> Zymo clean and concentrator, elute with 15 μL st.dH2O
|