TROUBLESHOOTING DNA AGAROSE GEL ELECTROPHORESIS

TROUBLESHOOTING DNA AGAROSE GEL ELECTROPHORESIS

If you see faint or no bands on the gel:

  • There was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don’t exceed 50 ng/band.
  • The DNA was degraded. Avoid nuclease contamination.
  • The DNA was electrophoresed off the gel. Electrophorese the gel for less time, use a lower voltage, or use a higher percent gel.
  • Improper W light source was used for visualization of ethidium bromide-stained DNA. Use a shortwavelength (254 nm) W light for greater sensitivity. Note: For preparative gels, using a longer wavelength (312 nm) W light will minimize DNA degradation.

If you see smeared DNA bands:

  • The DNA was degraded. Avoid nuclease contamination.
  • Too much DNA was loaded on the gel. Decrease the amount of DNA.
  • Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. This is done by checking the pH in the anode and cathode chambers.
  • There was too much salt in the DNA. Use ethanol precipitation to remove excess salts, prior to electrophoresis.
  • The DNA was contaminated with protein. Use phenol extractions to remove protein prior to electrophoresis.
  • Small DNA bands diffused during staining. Add the
    ethidium bromide during electrophoresis.

If you see anomalies DNA band migration:

  • Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity.
  • The DNA was denatured. Do not heat standards [except for l DNA/Hind III fragments (figure 1)] prior to electrophoresis. Dilute DNA standards in buffer with 20 mM NaCl.

FIGURE 1. The effect of ionic strength on the heat denaturation of l DNA/Hind III fragments. 500 ng DNA/Hind III fragments in NaCl concentrations of 0, 1, 2, 5, 10, 20, 30, and 40 mM were electrophoresed after heating at 65° C for 10 min.

Web source : http://www.bio.davidson.edu/courses/molbio/tips/trblDNAgel.html

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