User:Karmella Haynes/Notebook/PcTF Genomics/2013/01/25

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

--> 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

--> 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

--> 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

--> 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