Northern Blot: Glyoxal/DMSO

Objective:

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**	1.5uL	2	2.5uL	3uL
   *Freshly deionized (see text).
   **Add after heat denaturation (see text).

   
   
2. Mix the samples by tapping the end of the microfuge tube with your finger. Then briefly pulse the samples down in a microcentrifuge. Incubate at 50 degrees for one hour.
3. While the samples are incubating, prepare the gel: dissolve 0.8g agarose in 100mL of 10mM NaPO4 pH 7.0 (5mL of 0.2M NaP04 pH 7.0 + 95mL "DEPC" H₂O). Make sure gel box and comb are RNase-free by treatment with 3% H₂O₂ or commericial RNase removal product (e.g. RNase Zap from Ambion).
4. After samples are done incubating, place on ice and add loading buffer. Mix samples with pipette tip, and if necessary pulse down in microcentrifuge.
5. Place solidified gel in RNase-free gel box, and cover with 10mM NaPO4 pH 7.0 running buffer.
6. Load samples and run at 4V/cm until bromphenol blue has migrated approximately 8cms (approximately 3 hours). Recirculation of the running buffer (from the annode to the cathode) is required to prevent a H⁺ gradient and subsequent dissociation of glyoxal.
7. Remove gel.
8. To visualize, place all or portion of gel to be visualized in RNase-free dish and stain with 0.5ug/mL EtBr in 0.5M ammonium acetate for ~20-40 minutes. Then destain with 0.5M ammonium acetate for an additional ~20-40 minutes. Alternatively the ladder can be stained after gel transfer using methylene blue.

Reagents/Solutions

Preperation of 6M(40%) Glyoxal

• Deionize glyoxal with Bio-Rad AG 501-X8 (or X8D) resin until the pH is 5.0-5.5. Do this immediately before use, or after dionization, store in single-use aliquots (no freeze-thaw-freeze) in small tightly capped tubes.

  10X Loading Buffer

• 50% glycerol
• 10mM NaPO4 pH 7.0
• 0.25% bromophenol blue
• 0.25% xylene cyanol FF