Fine Purification of Probe DNA

Fine Purification of Probe DNA

  1. Bring appropriate amount of DNA of interest up to 315 µl TE in MFT.
  2. Phenol/CHCl3 extraction twice by adding 315 µl organic solvent, shake, spin (First spin, 12 K, 5 min; Second spin, 3 K, 5 min). Use yellow tips to transfer the top aqueous phase to another new MFT.
  3. CHCl3 extraction once (3K, 5 min)
  4. Ether extraction once (3K, 5 min). After take ether (anhydrous; diethyl ether, ethyl ether, or ethyl oxide; (C2H5H2O) fraction off from top with a yellow tip, open the vials to evaporate all ether (take 5 to 10 min).
  5. NH4Ac/EtOH precipitation (30 min/14 K): use cold EtOH.
    DNA suspension 315 µl
    NH4Ac 105 µl
    EtOH 1050 µl
  6. Rinse with 70 % cold EtOH twice, wipe, and dry 10 min under vacuum.
  7. Resuspend in appropriate amount (usually 10 µl) sterile milli-Q water.