Identification of plasmid DNA by agarose gel electrophoresis

Summary

Agarose is a linear polymer extracted from seaweed and should be electrophoretically pure. Agarose this grade screens out inhibitors and nucleases and has minimal fluorescent background when stained with ethidium bromide.

Operation method

electrophoresis

Materials and Instruments

Agarose
Ethidium bromide
Agarose gel electrophoresis device UV light

Move

1. Agarose gel electrophoresis apparatus
Because agarose gel electrophoresis is both undemanding and adaptable, a wide variety of electrophoresis tanks, large and small, have been successfully designed over the past 15 years. The choice of these units is largely based on personal preference. The most commonly used device is the horizontal plate gel invented by Walter Schaffner.
Horizontal plate gels are usually filled on a glass or plastic plate that can be placed on the platform of the electrophoresis tank. In some devices, the gel is laid directly on the platform. The gel is immersed just below the buffer level for electrophoresis. The resistance of the gel is almost the same as that of the buffer solution, so a significant portion of the current will pass through the full length of the gel.
2. Preparation of agarose gel
Agarose gels are prepared by melting agarose into a clear, transparent solution in the desired buffer. The melted solution is then poured into a gel mold and allowed to solidify. After solidification, the agarose forms a solid matrix whose density depends on the concentration of agarose. When the electric field through the gel is turned on, the negatively charged DNA at neutral pH migrates toward the anode.
3. Staining of the agarose gel
After electrophoresis, the agarose gel is transferred into a staining solution containing EB, stained for 10 minutes and removed for observation under UV light.


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Categories: Protocols