Detection of DNA staining in polyacrylamide gels

Summary

Unlike agarose gels, ethidium bromide cannot be added to polyacrylamide gels as this dye interferes with the polymerization of acrylamide. However, ethidium bromide can be used to stain polyacrylamide gels after electrophoresis. Since polyacrylamide will burst the fluorescence of ethidium bromide, the sensitivity of DNA detection is reduced. After electrophoresis, the polyacrylamide gel can also be stained with the nucleic acid dye SYBR Gold (Molecular Probes). This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.

Operation method

Detection of DNA staining in polyacrylamide gels

Principle

Unlike agarose gels, ethidium bromide cannot be added to polyacrylamide gels as this dye interferes with the polymerization of acrylamide. However, ethidium bromide can be used to stain polyacrylamide gels after electrophoresis. Since polyacrylamide will burst the fluorescence of ethidium bromide, the sensitivity of DNA detection is reduced. Polyacrylamide gels can also be stained with the nucleic acid dye SYBR Gold (Molecular Probes) after electrophoresis. It should not be incorporated into the gel during the polymerization process, as this will impede DNA migration and distort the DNA bands. Methylene blue staining is less sensitive but less expensive than the previous two dyes and can be used.

Materials and Instruments

Ethidium bromide solution SYBR Gold storage solution or methylene blue storage solution
Polyacrylamide gel

Move

I. Materials

1. Buffers and solutions

Dye solution: ethidium bromide solution (storage solution 0.5 μg/ml ), SYBR Gold storage solution or methylene blue storage solution (0.001%~0.0025%, TAE buffer preparation)

2. Gel

Polyacrylamide gel

II. Methods

1. Gently immerse the gel and the glass plate attached to it in the desired staining solution, just enough to cover the gel with the staining solution. Stain for 30-45 min at room temperature.

2. Remove the gel from the staining solution using the glass plate as a support, rinse with water, and dip the gel surface in a wad of Kimwipes tissue to remove excess liquid.

3. Cover the gel with a piece of Saran Warp film and use the wide end of a comb or a folded Kimwipe tissue to remove any air bubbles or wrinkles in the Saran Wrap film.

4. Place a piece of Saran Wrap membrane over a UV transmitted light source, invert the gel, place it over the transmitted light source, remove the glass plate, and leave the gel on the Saran Wrap membrane.

5. See protocol "Detection of DNA in Agarose Gels" for gel photography.

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