Isolation of RNA according to size: agarose gel electrophoresis of glyoxalized RNA

Summary

This method combines glyoxal denaturation and agarose gel electrophoresis (modified from that of McMaster and Carmichael (1977), Thomas (1983)). automated glyoxalization of RNA, ethidium bromide staining, and modification of electrophoresis buffer were provided by Burnett (1977). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Isolation of RNA according to size: agarose gel electrophoresis of glyoxalized RNA

Principle

This method combines glyoxal denaturation and agarose gel electrophoresis (modified from the method of McMaster and Carmichael (1977), Thomas (1983). automated glyoxalization of RNA, ethidium bromide staining, and modifications to the electrophoresis buffer were provided by Burnett (1977).

Materials and Instruments

RNA Sample RNA Size Standard Reference
BFTE Electrophoresis Buffer DMSO Ethylene Glycol Ethylene Glycol Reaction Mixture RNA Gel Sampling Buffer
Agarose Gel Horizontal Electrophoresis Unit Transparency Ruler Water Bath

Move

I. Materials

1. Buffers and solutions

10X BFTE electrophoresis buffer

DMSO

Ethylene Glycol

Glycol reaction mixture

RNA Gel Sampling Buffer

2. Gel

Agarose gel

3. nucleic acids and oligonucleotides

RNA Samples

RNA size standard reference

4. Specialized devices

Horizontal electrophoresis device

Transparent ruler

Water bath preset to 55°C

II. Methods

1. Prepare the glyoxal denaturing reaction solution. Mix in a sterile centrifuge tube:

RNA (10 μg total) 1~2 μl, ethylene glycol reaction buffer 10 μl.

2. Cap the centrifuge tube and place the RNA solution at 55°C for 60 min, ice bath in ice water for 10 min, and then centrifuge for 5 s to allow all liquid to settle to the bottom of the centrifuge tube.

3. Load the agarose gel into the box of the horizontal electrophoresis apparatus while the sample is heating. Add enough 1X BPTE Electrophoresis Buffer to cover the gel by about 1 mmol/L.

4. Add 1~2 μl of RNA Sampling Buffer to the glyoxalized RNA sample, and then immediately add the RNA sample to the gel spiking wells, leaving the two outermost wells on either side unspiked. Add the RNA relative molecular mass standard reference to the outermost well.

5. Electrophoresis is performed at 5 V/cm until the bromophenol blue migrates about 8 cm.

6. Place the gel on a piece of plastic wrap and observe the RNA under a UV light. align a transparent ruler with the gel and take a picture under the UV light.

7. Measure the distance from the spiking well to each RNA band on the photograph. Plot the migrated distances against the log ( _log10 ) value of the RNA fragment size. Use the resulting curve to calculate the size of the RNA detected by dot hybridization.

8. Immobilize the RNA on a solid support by upward or downward capillary transfer.

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