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Methods:Gel Electrophoresis

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Contents

Purpose

To separate analytes, whether nucleic acids or protein, based on size (1D) and Isoelectric point (2D).

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Materials Needed

  • Depends on the type of gel you intend to run, different analytes will require different gels.
  • Basic necessities:
    • Gel
    • Gel Electrophoresis apparatus(gel box and power source)
    • Analyte (DNA, RNA or protein)
    • Way to visualize your results after the gel is finished(staining)

Protocol

  • Make the gel solution and pour it into the gel block, insert gel comb so wells form
    • allow gel to solidify
  • Mix dye with the sample and standards, separate from stock solutions
    • most wells cannot contain volumes greater than 20μL, so 10μL of 2X Dye and 10μL or less of analyte (depending on analyte concentration)
      • Analyte concentration will determine band intensity, bands can be too intense.
  • remove the comb from the solidified gel, orient the gel properly in the gel block and add appropriate electrophoretic buffer until wells are submerged.
  • Inject analyte/dye solution into wells (write down what was injected into each well!)
  • Place lid on gel box, connect to power source, and run until dye front is near the bottom of gel
    • Do not let dye front run off the end of the gel, as this could result in loss of analyte
  • Remove gel carefully to avoid tearing the gel and use selected visualization technique.

Links

Wikibooks

Agarose gel for DNA

University of Utah interactive slide show

Wikipedia

User notes

2D gel visualization [1]

Way to improve visualization[2]

Molecular Weight[3]

Gel Gradients[4]

References

See Help:References for how to manage references in EcoliWiki.

  1. Van Belle, W et al. (2006) Adaptive contrast enhancement of two-dimensional electrophoretic protein gel images facilitates visualization, orientation and alignment. Electrophoresis 27 4086-95 PubMed EcoliWiki page
  2. Mohamed, MA et al. (1989) Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection. Anal. Biochem. 177 287-90 PubMed EcoliWiki page
  3. Weber, K & Osborn, M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244 4406-12 PubMed EcoliWiki page
  4. Muyzer, G & Smalla, K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73 127-41 PubMed EcoliWiki page