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Pc-TF Genomics
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01/25/13
- Illumina ChIP-seq prep: input DNA
Illumina ChIP-seq prep
> Follow manufacturer's protocol, with some modifications
> Sample (dated 12/04/10):
- "29, FTrx fx Input" (plain cells) -- this sample was used for the original ChIP-PCR experiments [1]
Perform End Repair
Note: forgot to dilute Klenow 1:5 with water before use
Reagent |
Volume
|
Input DNA |
30.0 μL
|
dH2O |
10.0
|
T4 DNA ligase buffer |
5.0
|
dNTP mix |
2.0
|
T4 DNA Polymerase |
1.0
|
Klenow DNA polymerase |
1.0
|
T4 PNK |
1.0
|
|
50 μL
|
--> 20°C/ 30 min. (PCR machine)
--> QIAquick PCR Purification, elute with 34 μL EB
Add 'A' Bases to the 3' End of the DNA Fragments
Reagent |
Volume
|
DNA sample |
34.0 μL
|
Klenow buffer |
5.0
|
dATP |
10.0
|
Klenow exo |
1.0
|
|
50 μL
|
--> Aliquot 16 μL master mix into DNA
--> 37°C/ 30 min. (heat block)
--> Zymo clean and concentrator, elute with 10 μL st.dH2O
Ligate Adapters to DNA Fragments
Note: Did not dilute the adapter oligo mix
Reagent |
Volume |
Master Mix
|
DNA sample |
10.0 μL |
---
|
DNA ligase buffer |
15.0 |
90.0
|
Adapter oligo mix |
1.0 |
6.0
|
DNA ligase |
4.0 |
24.0
|
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30 μL
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--> Aliquot 20 μL to DNA samples
--> 15 min./ r.t.
--> Zymo clean and concentrator, elute with 10 μL st.dH2O
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
|
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
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50.0 μL
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--> PCR machine (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
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