Athurva Gore/LabNotes/2011-4-8

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Bisulfite Biotin-Binding[edit]

  • Ran gel for Bisulfite-converted bead-bound DNA and normal bead-bound DNA:

File:04072011 Low Input Bisulfite FAILED.jpg

  • Bisulfite-converted libraries did not work.
    • DNA was definitely attached to beads, as normal amplification worked.
    • DNA must have been detached

Can we modify the experiment to still allow attachment of beads?[edit]

  • According to Invitrogen website [1], bead-detachment can occur when:
    • Heat in formamide or basic solution at 95 for 2 min or 65 for 5 min
    • Boil beads
    • Basic solutions, formamide, EDTA, and heat in general are bad
    • Specific detachment percentages: [2]
  • What could be causing detachment?
    • Bisulfite conversion involves adding an acidic solution with heat, incubation for a very long time
    • Desulphonation buffer is a strong base; could be detaching DNA
    • Other problems?
  • Possible solutions:
    • We can perform bisulfite conversion of the DNA first, and then attempt to bind beads
      • Potential pitfall: Bisulfite might not be a good buffer for binding, could inhibit bead-capture
      • Could we eliminate this problem by either
        • splitting each bisulfite reaction into multiple bead-binding reactions or
        • using less CT-conversion reagent (instead of 130 uL, use a lot less since we have much less DNA?)
      • One other commercial protocol uses IPure beads to capture bisulfite-converted DNA [3]
      • They just use a longer binding time; we can bind for 30 min to an hour to try
      • DO THIS TOMORROW
    • We can skip desulfonation step according to a published paper: [4]
      • Can use 30 min at 95 C as initial step in PCR; desulphonates DNA successfully without use of a base
      • Could be a big problem; heat could greatly damage DNA if exposed for that long