TROUBLESHOOTING DNA AGAROSE GEL ELECTROPHORESIS

Reproduced from Life Technologies, Inc.

Shanta Dube
Technical Services
Life Technologies, Inc.
Rockville, Maryland 20849

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.