User:Karmella Haynes/Notebook/PcTF Genomics/2013/01/25: Difference between revisions
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* | * Illumina ChIP-seq prep: input DNA | ||
---- | ---- | ||
''' | '''Illumina ChIP-seq prep'''<br> | ||
> | > Follow manufacturer's protocol, with some modifications<br> | ||
> Sample (dated 12/04/10): | |||
# "29, FTrx fx Input" (plain cells) -- this sample was used for the original ChIP-PCR experiments [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2011/04/22] | |||
<br> | |||
<u>Perform End Repair</u><br> | |||
Note: forgot to dilute Klenow 1:5 with water before use | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | ||
|-valign="top" | |-valign="top" | ||
| <u>Reagent</u> || <u>Volume</u> | | <u>Reagent</u> || <u>Volume</u> | ||
|- | |||
| Input DNA || 30.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 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)<br> | |||
--> QIAquick PCR Purification, elute with 34 μL EB | |||
<u>Add 'A' Bases to the 3' End of the DNA Fragments</u><br> | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | |||
|-valign="top" | |||
| <u>Reagent</u> || <u>Volume</u> | |||
|- | |- | ||
| | | 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<br> | ||
--> 37°C/ 30 min. (heat block)<br> | |||
--> Zymo clean and concentrator, elute with 10 μL st.dH<sub>2</sub>O | |||
<u>Ligate Adapters to DNA Fragments</u><br> | |||
Note: Did not dilute the adapter oligo mix<br> | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | |||
|-valign="top" | |||
| <u>Reagent</u> || <u>Volume</u> | |||
|- | |||
| DNA sample || 10.0 μL | |||
|- | |||
| DNA ligase buffer || 15.0 | |||
|- | |||
| Adapter oligo mix || 1.0 | |||
|- | |||
| DNA ligase || 4.0 | |||
|- | |||
| || 30 μL | |||
|} | |||
--> Aliquot 20 μL to DNA samples<br> | |||
--> 15 min./ r.t.<br> | |||
--> Zymo clean and concentrator, elute with 10 μL st.dH<sub>2</sub>O<br> | |||
--> Store at -20°C | |||
---- | |||
Day 2 (1/26/13)<br> | |||
<u>Size Select the Library</u><br> | |||
> Run all samples (+ loading buffer) in a 2% TAE gel<br> | |||
> Use 100 bp ladder to excise area from 150 - 250<br> | |||
> Purify DNA with a Zymoclean Gel DNA recovery kit<br> | |||
> Elution: 2x 10 μL, add 16 μL st.dH<sub>2</sub>O to eluate (36 μL total) | |||
[[Image:KAH012613_gel1.tif|300px|Gel purification 01/26/13]] | |||
<u>Enrich the Adapter-Modified DNA Fragments by PCR</u><br> | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table --> | |||
|-valign="top" | |||
| <u>Reagent</u> || <u>Volume</u> || <u>Master mix (6x)</u> | |||
|- | |||
| 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 | |||
|- | |||
| || 50.0 μL | |||
|} | |||
--> PCR machine (heat lid 100°C):<br> | |||
* 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.dH<sub>2</sub>O | |||
Revision as of 18:45, 26 January 2013
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01/25/13
Illumina ChIP-seq prep
Perform End Repair
--> 20°C/ 30 min. (PCR machine)
--> Aliquot 16 μL master mix into DNA
--> Aliquot 20 μL to DNA samples Day 2 (1/26/13) Size Select the Library
--> PCR machine (heat lid 100°C):
--> Zymo clean and concentrator, elute with 15 μL st.dH2O
|