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        Northern Blot: Glyoxal/DMSO

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        Northern Blot: Glyoxal/DMSO

        Adapted from Molecular Cloning (Maniatis), Current Protocols in Molecular Biology, and J. Yost Lab (UMN)

        Objective:

        Glyoxal/DMSO gels are great for determining sizes of RNA especially in conjunction with northern blots.

         

        Procedures:

          Start=> RNA, either isolated total RNA, poly(A+ )RNA, or in vitro transcribed RNA.

        1. Begin by preparing the sample for heat denaturation. Combine all of the reagents from the following table except 10X loading buffer in microcentrifuge tubes. If many samples are being prepared, it may be more efficient to make a master mix of NaPO4, DMSO, and glyoxal. To make the master mix, multiply the amount of each component required per sample by the number of samples + 10% (don't forget the ladder). The choice of sample volume depends on well-size and RNA concentration.

          Preperation of RNA sample
          Reagent For 15uL For 20uL For 25uL For 30uL
          RNA (up to 10ug) 3uL 4uL 5uL 6uL
          0.2M NaPO4 pH 7.0 0.75uL 1uL 1.25uL 1.5uL
          DMSO 7.5uL 10uL 12.5uL 15uL
          6M(40%) Glyoxal 3uL 3.75uL 2.25uL 4.5uL
          10X Loading Buffer Freshly deionized (see text).
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