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Oligonucleotide Purification

Method

Pre-siliconize plates and one Eppendorf tube per oligo with Sigmacote®.

  1. Resuspend (or dry down) oligo to 33 µg/µl (OD260) in H2O.
  2. Take 8 µl 33 µg/µl oligo + 8 µl formamide and heat at 90°C for 2 minutes.

    If you need more than 33 µg of oligo, run more than one lane.
  3. Make a medium-sized 15% PAGE sequencing gel. Pre-run gel for ~15 minutes, and then flush wells. Run a separate lane with Sequencing Stop Buffer, which contains bromophenol blue (BMB) and xylene cyanol (XC) for sizes. BMB runs approximately 10 bases and XC runs approximately 55 bases.
  4. Run at 300V for ~2-4 hours.
  5. Remove top gel plate and cover gel with plastic wrap, then flip and coax gel off of bottom plate. Then cover other side with plastic wrap.
  6. Place gel on intensifying screen and look from the top with shortwave UV (minimal time). You should see an intense band corresponding to your oligo plus a smear of smaller, incompletely synthesized DNA.

    Notes: If you overload the lanes, you will not get good separation and will end up having a lot of contamination in the "intense band" that you see in an overloaded lane.

    Use a marker to circle area on one side of plastic wrap.
  7. Remove from UV, flip gel over and remove plastic wrap from other side. Cut out indicated area of gel, dice up gel fragment and place in siliconized Eppendorf.
  8. Add 1ml 0.5M NH4Ac/0.01M Mg(Ac)2. Vortex and incubate 6 hours O/N at 37°C.
  9. Spin down gel pieces and withdraw supernatant. Rinse gel slices with an additional 0.5 ml same buffer and add to other supernatant.
  10. Dry down in SpeedVac to about 200 µl. Then precipitate DNA with 1 ml 100% ethanol (The salts will probably precipitate but should dissolve during the ethanol washes.).
  11. Wash pellet (and salts) with 70% ethanol and spin down DNA.
  12. Resuspend DNA in 200 µl H2O.
  13. Extract with phenol:chloroform twice.
  14. Read at OD260 of 5 µl in 100 µl for concentration.

    15% Gel (0.5-mm thickness)

    • 31.5 g Urea
    • 29.5 ml Acrylamide (40%, 19:1)
    • 15 ml 5X TBE
    • 8 ml H2O

*From S. Y. Roth Dent: Personal notes 1999


© 2014 The University of Texas MD Anderson Cancer Center