Athurva Gore/LabNotes/2011-4-8
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Bisulfite Biotin-Binding
- 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 to beads?
- 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 skip desulfonation step according to a published paper: [3]
- Can use 30 min at 95 C as initial step in PCR; desulphonates DNA successfully without use of a base
- We can skip desulfonation step according to a published paper: [3]