Dinh/Dinh 2015/NOTES/2015-9-20
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oxBS for H1 ESC p48[edit]
AMPure beads purification on H1 ESCs DNA + controls[edit]
- Set up (final volume = 50 uL):
H1 ESC gDNA (117 ng/uL) 17.94 uL (2.1 ug) 5hmC/5mC/C controls: 1.29 ul (21 ng) Nuclease free water: 30.00 uL
- Add 100 uL AMPure XP beads to 50 uL of DNA mixture.
- Incubate for 10 minutes.
- Wash 3X with 80% acetonitrile, I disturbed beads gently to get cleaner washes.
- Dry for 5 minutes at room temperature until beads are cracked.
- Elute with 80 uL nuclease free water.
- Incubate beads in water for 10 minutes at 55C.
- QUBIT: 14.5 ng/uL x 80 uL = 1160 ng (55% yield).
Prepare and test oxidant[edit]
- Prepare 20 mM oxidant (total = 20 uL):
108 mM KRuO4 3.70 uL 1 M NaOH 1.00 uL Water 15.30 uL
- Test DNA mixture purity:
1 uL purified DNA 4 uL water 1 uL 20 mM oxidant mixture
- Confirm that the solution did not immediately turned dark.
Denaturation[edit]
- Prepare 2 replicates each of oxBS, and BS samples, left ~4 uL of un treated DNA as a control.
- Prepare reactions in 2.0 mL round bottom tubes.
- Set up (final volume is 24 uL):
Purified DNA 18.00 uL 1 M NaOH 1.25 uL Water 5.75 uL
- Vortex briefly and spin down.
- Incubate on thermomixer at 37C for 30 minutes x 650 rpm
Oxidation[edit]
- Add 2 uL of 20 mM KRuO4 mixture to the two oxBS replicates.
- Add 2 uL of water to the two BS replicates.
- Do nothing to the no treatment control.
- Incubate on thermomixer at 40C for 15 minutes x 650 rpm
Epitect bisulfite conversion[edit]
- Set up bisulfite reactions for oxBS and BS samples (final volume is 140 uL):
Treated DNA 26 uL DNA protect buffer 29 uL Bisulfite mix 85 uL
- Do nothing to the no treatment control.
- Incubate in the thermocycler O/N
- No carrier RNA added during purification.
- Qubit:
oxBS1 9.22 ng/uL x 20 = 184 ng (70.6% yield) oxBS2 7.03 ng/uL x 20 = 141 ng (53.9%) BS1 11.10 ng/uL x 20 = 222 ng (85.0%) BS2 8.17 ng/uL x 20 = 163 ng (62.6%)
Control DNA preparation[edit]
- See [[1]]