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œœœœœ@Glyoxal gels@œœœœœ



RNA can be completely denatured by glyoxalating the bases in the presence of DMSO, thus prohibiting base pairing. RNAs denatured in this way will migrate in agarose or PAGE gels without interference because of secondary structure. Therefore, with this system, the molecular weights of RNAs can be closely estimated by comparing them to known standards (usually glyoxalated E.coli RNA) without the use of methylmercuric hydroxide. However, samples treated in this way should be relatively 'clean', so that the glyoxal is not depleted by contaminanting junk.
Strict RNA-free technique should be used during this technique. All buffers, plasticware, and glassware should be autoclaved before use (except DMSO and glyoxal), and the electrophoresis comb and tray should be cleaned with EtOH. DON'T TOUCH THE TEETH ON THE COMB! If desired, 15mM Na iodoacetate can be incorporated into the gel.

  1. Materials

    1. 0.2M NaH2PO4
      pH 7.0 - 2.76 grams/100ml & pH with 5M NaOH

    2. 7M Glyoxal
      Glyoxal is usually supplied at 7M. Before use, deionize by stirring with 10g mixed resin ion exchanger per 20ml, and store in small aliquots at -20C.

    3. 100X GE buffer (per liter)
      Final Conc.
      69.0 grams NaH2PO4 0.5M
      134 grams Na2HPO4-7H2O 0.5M - 1M PO4 total
      1 liter
      Adjust to pH 7.0 with 50% NaOH
    4. GE
      diluted from 100X stock (10ml/l)

    5. Acridine orange stain
      Final Conc.
      5 mg acridine orange 10ug/ml
      0.2922 grams NaCl 10mM
      500ml

  2. Sample preparation:

    1. Mix the following in a 500ul microfuge tube:
      25ul DMSO (optional, if more RNA volume is needed)
      2.5ul 0.2M NaH2PO4, pH 7.0
      7.1ul 7M glyoxal
      15.3ul RNA sample
      50ul total volume

    2. Incubate capped at 50C for 1 hr.
    3. Add 5ul 10X tracking dye and load into the gel.

  3. Gel:

    1. Use a 1-2.5% agarose gel (or 5-12% PAGE) made in GE. The gel should be poured while hot - about85C. 15mM Na iodoacetate can be added if RNase activity in the gel is a problem.
    2. Recirculate the buffer by running the gel with the buffer trays on top of stir-motors, with gentle stirring, and with a peristaltic pump exchanging buffer between the trays.
    3. Electrophorese at 1-5 V/cm until the dye reaches about 3/4 of the way down the gel.
    4. Stain the gel for 1 hr in acridine orange, then destain by soaking in ddH2O for 1-1.5 hr. Examine under a UV light. To photograph, use Kodak Wratten filters; #12 for both green and red bands, #40 for green bands (DNA),and #29 for red bands (RNA).


McMaster and Carmichael 1977 PNAS USA 74:4835
Maniatis, et al 1982 "Molecular Cloning" CSH
Davis, et al 1980 "Advanced Bacterial Genetics" CSH pp156-158

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