Under slightly acidic conditions, the two acetaldehyde groups of glyoxal interact with the imino group of guanosine to form a cyclic compound, which inhibits the formation of the interchain Watson-Crick bond and prevents the RNA from forming a stable secondary structure.Its mobility in agarose gel electrophoresis is directly proportional to the logarithm of its size.

Operation method

Glyoxal/DMSO denaturing agarose gel electrophoresis

Materials and Instruments

10XBPTE Electrophoresis Buffer Dimethyl Sulfoxide (DMSO) Deionized Glyoxal Glyoxal Antifu Mix Sampling Buffer
Horizontal Electrophoresis Unit

Move

(i) Village materials and equipment

1) 10XBPTE electrophoresis buffer: 100 mmol/LPIPES, 300 mmol/LTris, lOmmol/LEDTA (pH 8.0)

2) Dimethyl sulfoxide (DMSO)

3) Deionized glyoxal

4) Ethanal antifugal mixture: 6 ml DMSO, 2 ml deionized ethanedial, 2 mL 10XBPTE electrophoresis buffer, 0.6 ml 80% glycerol, 0.2 ml ethidium bromide (10 mg/ml).

5) Sampling buffer: 50% glycerol, 10 mmol/L sodium phosphate (PH7.0), 0.25% bromophenol blue, 0.25% xylene cyanide.

6) Horizontal electrophoresis unit


(ii) Methods of operation

1) Preparation of gel: Prepare 1.2% agarose gel with 1XBPTE electrophoresis buffer.

2) Electrophoresis: Add 10ul of glyoxal reaction mixture to 1~2ul of RNA sample (10ug in total), keep warm at 55℃ for lh, ice bath for 10 min, centrifuge, add 1~2ul of sampling buffer, and then electrophoreze the samples immediately. 1XBPTE was used as the electrophoresis buffer and electrophoresis was performed at a constant pressure of 60℃, and the electrophoresis was stopped when the migration of bromophenol blue was 1/2~2/3.

3) Observation: Observe the results under UV light.

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