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
- We can perform bisulfite conversion of the DNA first, and then attempt to bind beads