Nucleic Acid Stains, Ladders, and Markers Support—Troubleshooting
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Nucleic Acid Stains
There are two things that affect a DNA band’s migration as it travels through the gel matrix.
- Dye charge. DNA migrates through the matrix due to its negative charge, causing it to migrate towards the anode. However, the fluorescent dyes that bind to the DNA have their own charge and can either enhance or hinder (the most common) the migration rate.
- Dye affinity. Some dyes have a high affinity for DNA and cause a significant change in the migration rate. However, most dyes have a level of affinity that results in the dye effectively binding and dissociating from the DNA molecule at some rate. It is this event that can cause the most problems during electrophoresis. If the dye dissociates from the DNA and there is not a ready replacement (due to prestaining the DNA prior to loading, for example where there is no dye in the gel itself), then as the DNA is running, it will have a decreasing amount of dye bound to it. When this happens, you will usually see band broadening and possibly a change in migration pattern. This effect usually disturbs lower molecular weight bands more, as usually there is less mass per band at lower molecular weights, which means there is a lower total amount of dye to begin with in that band. This is why we usually recommend poststaining with most of the SYBR® dyes and either prestaining the entire gel or poststaining with SYBR® Safe DNA Gel Stain. Some people have found that increasing the concentration of the SYBR® dye in the loading buffer to 10X (e.g., performing a 1:1,000 dilution from the normal 10,000X working stock) reduces these effects.
Many whitening agents used in clothing, as well as some fungi and bacteria, fluoresce at the same wavelengths as SYBR® Safe DNA gel stain. These contaminants within or on the surface of the gel may produce this speckling.
Nucleic Acid Ladders and Markers
This can happen if the marker was heated. Please ensure that the ladders are not heated before use.
Here are a few reasons why you might see smearing of the bands:
- The DNA was degraded. Avoid nuclease contamination of DNA standards.
- Too much DNA was loaded on the gel. Decrease the amount of DNA in the gel.
- The DNA was contaminated by protein. Remove proteins by phenol extraction before electrophoresis.
- For small DNA, the bands may have diffused during staining. Add the ethidium bromide before electrophoresis.
- For the Supercoiled DNA Ladder, insufficient ethidium bromide was used. Verify that the samples contain 10–50 μg/mL final concentration of ethidium bromide.
- For radiolabeled DNA, labeling was performed by nick translation. Label the DNA by replacement synthesis with T4 DNA polymerase or label the 5' end with T4 polynucleotide kinase.
- 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 has sufficient buffering capacity.
- The DNA contained too much salt. Remove excess salt by ethanol precipitation before electrophoresis.
There are a few possible reasons for this:
- Small DNA bands were electrophoresed off the gel. Electrophorese the gel for less time or at lower voltage, or use a higher percentage gel.
- The small bands migrated with the dye front due to differences in ionic strength between gel and buffer. Be sure the buffer in the gel is the same as the electrophoresis buffer.
- DNA bands of similar molecular size were not resolved. Increase the electrophoresis time and check the proper percentage gel for resolution. For DNA fragments <1,000 bp, try Agarose 1000 (Cat. No. 16550100) instead of agarose.
- The DNA was denatured. Do not heat standards (except lambda phage-derived markers) prior to electrophoresis. Dilute markers in a buffer containing 20 mM NaCl.
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