User:Karmella Haynes/Notebook/PcTF Genomics/2011/12/14

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12/14/11

  • ✓ Illumina ChIp-seq prep: Pc-TF ChIP samples 1-6 (Day two)


Illumina ChIp-seq prep
> See 12/13/11 for list of samples (1-6)

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 12/14/11


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

--> Xiao's PCR machine, block A (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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