AlanFung:LabNotes/CTCF/2011-7-28

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Low-input Bisulfite Conversion[edit]

  • I will be working on the bisulfite conversion first protocol.

Protocols Today[edit]

  • Start with four tubes: 1571 cells, 314 cells, 157 cells, NTC, and cells that do not go through bisulfite conversion

Cell Lysis[edit]

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
    • Add 1 uL Protease to 99 uL H2O for a 1:100 dilution
  • Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Cells are now lysed

Bisulfite Conversion - Imprint Kit[edit]

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 21 uL 0.5 mg/mL BSA and 1 uL Imprint Balance solution to each sample; incubate at 37 C for 10 minutes
  • Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
  • Perform Column Purification of each sample:
    • Elute into 10 uL Elution Buffer

MDA - Epitect Kit[edit]

  • Denature the no bisulfite conversion control by heating at 95C for 30 sec and cool down immediately with a cold block
  • Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly.
  • Add 1ul REPLI-g Midi DNA Polymerase to 29ul WBA reaction buffer
  • Mix and centrifuge briefly
  • Add 30ul Master mix to 10ul of bisulfite converted DNA
  • Incubate at 28C for 1 h and 95C for 5 min

MinElute[edit]

  • Elute with 12ul buffer

Nanodrop[edit]

  • Perform nanodrop on a few of the samples
  • 1500cells-12.9ng/ul
  • 150cells(NBS)-31.8ng/ul
  • 1500cells(NBS)-48ng/ul

Tagmentation - Shendure Protocol[edit]

  • Take 3ul of the MDA product for tagmentation
  • To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
  • Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Store at -20C

Amplification - Kapa + BST[edit]

Content Volume MM
Nuclease Free H2O 16 140.8
10uM Orange Primer 1 8.8
10uM Blue Primer 1 8.8
Tagmented DNA 6 Do not add to MM
2X Kapa sybr supermix 25 220
BST 1 8.8
Total 50 387.2
  • Transfer DNA to strip tube and add 44ul MM to each reaction
  • Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O)
  • Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample
  • Perform qPCR using the following reaction conditions:
    • 60 C, 20 min incubation (BST second strand synthesis)
    • 72 C, 3 min incubation (Nextera extension (where necessary; not needed if using Klenow))
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extend)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C

PAGE Gel Analysis[edit]

File:ZhangLab 2 2011-07-29 14hr 42min.jpg

Results[edit]

File:072911.jpg

  • Shouldn't have heat inactivates the MDA product as that will denature the DNA and makes it single stranded.
  • I can probably skip the inactivation step and just perform a column purification to eliminate the polymerase