Troubleshooting DNA Separations on Agarose Gels

 

Problem

Possible Cause

Suggested solution

Faint or no DNA bands Insufficient quantity of concentration of DNA was loaded on gel. Increase the amount of DNA. Notes: A low concentration may be due to volume added per well width.
DNA was degraded. Avoid nuclease contamination of DNA.
DNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel.
For ethidium bromide-stained DNA, improper UV light source was used. Use short-wavelength (254 nm) UV light for greater sensitivity.
Missing DNA bands Small DNA bands were electrophoresed off the gel.

Electrophorese the gel for less time, at lower voltage, or in a higher percentage gel.

Be sure buffer in the gel is the same as the electrophoresis buffer.

DNA bands of similar molecular size were not resolved. Increase the electrophoresis time (lower voltage may be required) and check the proper percentage gel for resolution.
DNA was denatured. Do not heat standards (except lambda DNA fragments) prior to electrophoresis
Smeared DNA bands DNA was degraded. Avoid nuclease contamination of DNA.
Too much DNA was loaded on gel. Decrease the amount of DNA in the gel.
DNA contained too much salt. Remove excess salt by ethanol precipitation before electrophoresis.
DNA was contaminated by protein. Remove proteins by phenol extraction before electrophoresis.
For small DNA, the bands diffused during staining. Add ethidium bromide during electrophoresis.
DNA was denatured. Do not heat DNA prior to electrophoresis. Dilute DNA in buffer with 20 mM NaCl.
For the supercoiled DNA, insufficient DNA was used. Verify that samples contain 2 µg/ml of ethidium bromide.
Anomalous DNA band migration Improper electrophoresis conditions were used.

Do not allow voltage to exceed 20 V/cm.

Maintain temperature of <30°C during electrophoresis.

Check that the electrophoresis buffer used has sufficient buffering capacity.

DNA was denatured. Do not heat DNA prior to electrophoresis. Dilute DNA in buffer with 20 mM NaCl.
Little difference in band intensity when using quantitation standards on ethidium bromide-stained gels. Excess volume loaded on gel. The smallest loading volume will result in the greatest difference in band thickness between different masses.
Sample loaded in larger or smaller volume than standard. Load equal volumes of sample and standard DNA for accurate comparison.
Ethidium bromide staining after electrophoresis. Include ethidium bromide in the gel rather than staining after electrophoresis to increase the difference in band intensity.
Curved bands Unequal temperature across gel. Cool entire gel equally, or not at all.
Unequal buffer concentration between gel and electrophoresis buffer. Make gel buffer and electrophoresis buffer together in same batch.