Difference between revisions of "User:Karmella Haynes/Notebook/PcTF Genomics/2013/01/25"

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(Autocreate 2013/01/25 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
 
(mm/dd/yy)
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==mm/dd/yy==
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==01/25/13==
 
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* Line item 1
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* Illumina ChIP-seq prep: input DNA
 +
 
  
 
----
 
----
'''Line item 1'''<br>
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'''Illumina ChIP-seq prep'''<br>
> Samples
+
> 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 || ---
 
|-
 
|-
| reagent 1 || # 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
 +
|-
 +
| &nbsp; || 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>
 
|-
 
|-
| reagent 2 || #
+
| DNA sample || 34.0 μL || ---
 
|-
 
|-
| reagent 3 || #
+
| Klenow buffer || 5.0 || 30.0
 
|-
 
|-
| reagent 4 || #
+
| dATP || 10.0 || 60.0
 
|-
 
|-
| dH<sub>2</sub>O || #
+
| Klenow exo || 1.0 || 6.0
 
|-
 
|-
| &nbsp; || # μL
+
| &nbsp; || 50 μL
 
|}
 
|}
  
--> Reaction conditions
+
--> 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
 +
|-
 +
| &nbsp; || 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 13:40, 25 January 2013

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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):

  1. "29, FTrx fx Input" (plain cells)


Perform End Repair
Note: forgot to dilute Klenow 1:5 with water before use

Reagent Volume Master Mix
Input DNA 30.0 μL ---
dH2O 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
--> 20°C/ 30 min. (temp block)
--> QIAquick PCR Purification, elute with 34 μL EB


Add 'A' Bases to the 3' End of the DNA Fragments

Reagent Volume Master Mix
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
--> 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
  30 μL

--> Aliquot 20 μL to DNA samples
--> 15 min./ r.t.
--> Zymo clean and concentrator, elute with 10 μL st.dH2O
--> Store at 4°C o/n