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) | |||
<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> || <u>Master Mix</u> | ||
|- | |||
| Input DNA || 30.0 μL || --- | |||
|- | |- | ||
| | | dH<sub>2</sub>O || 10.0 || 60.0 | ||
|- | |||
| T4 DNA ligase buffer || 5.0 || 30.0 | |||
|- | |||
| dNTP mix || 2.0 || 12.0 | |||
|- | |||
| T4 DNA Polymerase || 1.0 || 6.0 | |||
|- | |||
| Klenow DNA polymerase || 1.0 || 6.0 | |||
|- | |||
| T4 PNK || 1.0 || 6.0 | |||
|- | |||
| || 50 μL | |||
|} | |||
--> Aliquot 20 μL master mix, add 30 μL ChIP DNA<br> | |||
--> 20°C/ 30 min. (temp block)<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> || <u>Master Mix</u> | |||
|- | |- | ||
| | | DNA sample || 34.0 μL || --- | ||
|- | |- | ||
| | | Klenow buffer || 5.0 || 30.0 | ||
|- | |- | ||
| | | dATP || 10.0 || 60.0 | ||
|- | |- | ||
| | | Klenow exo || 1.0 || 6.0 | ||
|- | |- | ||
| || | | || 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> || <u>Master Mix</u> | |||
|- | |||
| 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 | |||
|- | |||
| || 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 4°C o/n | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 14:40, 25 January 2013
Pc-TF Genomics | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/25/13
Illumina ChIP-seq prep
Perform End Repair
--> Aliquot 20 μL master mix, add 30 μL ChIP DNA
--> Aliquot 16 μL master mix into DNA
--> Aliquot 20 μL to DNA samples |