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.