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)
Current revision (21:45, 26 January 2013) (view source)
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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
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----
----
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'''Line item 1'''<br>
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'''Illumina ChIP-seq prep'''<br>
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> Samples
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> Follow manufacturer's protocol, with some modifications<br>
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> Sample (dated 12/04/10):
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# "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]
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<br>
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<u>Perform End Repair</u><br>
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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>  
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| <u>Reagent</u> || <u>Volume</u>
|-
|-
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| reagent 1 || # μL
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| Input DNA || 30.0 μL  
|-
|-
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| reagent 2 || #
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| dH<sub>2</sub>O || 10.0
|-
|-
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| reagent 3 || #
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| T4 DNA ligase buffer || 5.0
|-
|-
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| reagent 4 || #
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| dNTP mix || 2.0
|-
|-
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| dH<sub>2</sub>O || #
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| T4 DNA Polymerase || 1.0
|-
|-
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| &nbsp; || # μL
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| Klenow DNA polymerase || 1.0
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|-
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| T4 PNK || 1.0
 +
|-
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| &nbsp; || 50 μL
|}
|}
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--> Reaction conditions
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--> 20°C/ 30 min. (PCR machine)<br>
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--> QIAquick PCR Purification, elute with 34 μL EB
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 +
 
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<u>Add 'A' Bases to the 3' End of the DNA Fragments</u><br>
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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
 +
|-valign="top"
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| <u>Reagent</u> || <u>Volume</u>
 +
|-
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| DNA sample || 34.0 μL
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|-
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| Klenow buffer || 5.0
 +
|-
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| dATP || 10.0
 +
|-
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| Klenow exo || 1.0
 +
|-
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| &nbsp; || 50 μL
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|}
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--> 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>
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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>
 +
|-
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| DNA sample || 10.0 μL
 +
|-
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| DNA ligase buffer || 15.0
 +
|-
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| Adapter oligo mix || 1.0
 +
|-
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| DNA ligase || 4.0
 +
|-
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| &nbsp; || 30 μL
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|}
 +
 
 +
--> Aliquot 20 μL to DNA samples<br>
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--> 15 min./ r.t.<br>
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--> Zymo clean and concentrator, elute with 10 μL st.dH<sub>2</sub>O<br>
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--> Store at -20°C
 +
 
 +
----
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Day 2 (1/26/13)<br>
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<u>Size Select the Library</u><br>
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> Run all samples (+ loading buffer) in a 2% TAE gel<br>
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> Use 100 bp ladder to excise area from 150 - 250<br>
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> 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]]
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 +
 
 +
<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>
 +
|-
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| DNA || 36.0 μL || ---
 +
|-
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| 5x Phusion buffer || 10.0 || 60.0
 +
|-
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| dNTP mix || 1.5 || 9.0
 +
|-
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| PCR primer 1.1 || 1.0 || 6.0
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|-
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| PCR primer 2.1 || 1.0 || 6.0
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|-
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| Phusion polymerase || 0.5 || 3.0
 +
|-
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| &nbsp; || 50.0 μL
 +
|}
 +
 
 +
--> PCR machine (heat lid 100°C):<br>
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* 98°C, 30 sec
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* 18x [98°C, 10 sec/ 65°C, 30 sec/ 72°C, 30 sec]
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* 72°C, 5 min
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* 4°C, ∞
 +
--> Zymo clean and concentrator, elute with 15 μL st.dH<sub>2</sub>O
 +
 

Current revision

Pc-TF Genomics Main project page
Previous entry      


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)

Image:KAH012613 gel1.tif


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




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