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

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{| 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>Master Mix</u>
+
| <u>Reagent</u> || <u>Volume</u>
 
|-
 
|-
| Input DNA || 30.0 μL || ---
+
| Input DNA || 30.0 μL  
 
|-
 
|-
| dH<sub>2</sub>O || 10.0 || 60.0
+
| dH<sub>2</sub>O || 10.0
 
|-
 
|-
| T4 DNA ligase buffer || 5.0 || 30.0
+
| T4 DNA ligase buffer || 5.0  
 
|-
 
|-
| dNTP mix || 2.0 || 12.0
+
| dNTP mix || 2.0  
 
|-
 
|-
| T4 DNA Polymerase || 1.0 || 6.0
+
| T4 DNA Polymerase || 1.0  
 
|-
 
|-
| Klenow DNA polymerase || 1.0 || 6.0
+
| Klenow DNA polymerase || 1.0  
 
|-
 
|-
| T4 PNK || 1.0 || 6.0
+
| T4 PNK || 1.0  
 
|-
 
|-
 
| &nbsp; || 50 μL
 
| &nbsp; || 50 μL
 
|}
 
|}
  
--> Aliquot 20 μL master mix, add 30 μL ChIP DNA<br>
+
--> 20°C/ 30 min. (PCR machine)<br>
--> 20°C/ 30 min. (temp block)<br>
 
 
--> QIAquick PCR Purification, elute with 34 μL EB
 
--> QIAquick PCR Purification, elute with 34 μL EB
  
Line 53: Line 52:
 
{| 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>Master Mix</u>
+
| <u>Reagent</u> || <u>Volume</u>
 
|-
 
|-
| DNA sample || 34.0 μL || ---
+
| DNA sample || 34.0 μL
 
|-
 
|-
| Klenow buffer || 5.0 || 30.0
+
| Klenow buffer || 5.0  
 
|-
 
|-
| dATP || 10.0 || 60.0
+
| dATP || 10.0
 
|-
 
|-
| Klenow exo || 1.0 || 6.0
+
| Klenow exo || 1.0  
 
|-
 
|-
 
| &nbsp; || 50 μL
 
| &nbsp; || 50 μL
Line 76: Line 75:
 
{| 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>Master Mix</u>
+
| <u>Reagent</u> || <u>Volume</u>
 
|-
 
|-
| DNA sample || 10.0 μL || ---
+
| DNA sample || 10.0 μL
 
|-
 
|-
| DNA ligase buffer || 15.0 || 90.0
+
| DNA ligase buffer || 15.0  
 
|-
 
|-
| Adapter oligo mix || 1.0 || 6.0
+
| Adapter oligo mix || 1.0  
 
|-
 
|-
| DNA ligase || 4.0 || 24.0
+
| DNA ligase || 4.0  
 
|-
 
|-
 
| &nbsp; || 30 μL
 
| &nbsp; || 30 μL
Line 92: Line 91:
 
--> 15 min./ r.t.<br>
 
--> 15 min./ r.t.<br>
 
--> Zymo clean and concentrator, elute with 10 μL st.dH<sub>2</sub>O<br>
 
--> Zymo clean and concentrator, elute with 10 μL st.dH<sub>2</sub>O<br>
--> Store at 4°C o/n
+
--> 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
 +
|-
 +
| &nbsp; || 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
 +
 
 +
 
 +
 
  
 
<!-- ##### 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. ##### -->

Latest revision as of 18:45, 26 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) -- 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
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
--> 15 min./ r.t.
--> Zymo clean and concentrator, elute with 10 μL st.dH2O
--> Store at -20°C


Day 2 (1/26/13)

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)

Gel purification 01/26/13


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
  50.0 μL

--> 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