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

From OpenWetWare
Jump to navigationJump to search
Line 17: Line 17:
> Follow manufacturer's protocol, with some modifications<br>
> Follow manufacturer's protocol, with some modifications<br>
> Sample (dated 12/04/10):
> 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)
# "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>
<br>


Revision as of 14:50, 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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>


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 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


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/01/25&oldid=670608"