Northern Blot: Glyoxal/DMSO

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

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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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