Dinh/Dinh 2015/NOTES/2015-9-20

From ZhangLabWiki
Jump to navigation Jump to search

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]