Gel electrophoresis is the technique where DNA molecules move through the agarose gel, based on their different mass to charge ratio.
We offer great range of agarose to choose from, please click here >.
The word "phoresis" means movement or migration and "electro" means electricity, which means movement with the help of electricity. Based on their size and charge, the molecules will travel through the agarose gel at different speeds, allowing them to be separated from one another. The size of the separated fragment is then determined by examining it next to the DNA ladder containing DNA fragments of specified different sizes with the help of UV light. As easy as it may sound, it is an art in itself which sometimes require a lot of troubleshooting to get to the results.
So we made a troubleshooting guide only for you!!
| Problem | Causes | Recommended measures |
| No or light DNA bands |
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| Smiley bands | Excess use of intercalating dye | • GelRed is very sensitive and requires addition of very less PCR product as compared to when EtBr is used. Using more than recommended intercalating dye may change the conformation of DNA plasmid leading to its different migrant speed |
| Smeared bands | Incorrect gel type | • Use a denaturing gel for SSRNA and not for DNA samples |
| Too much salt | • Remove the excess salt via ethanol precipitation before electrophoresis | |
| Protein contamination | • Remove all protein using phenol extraction | |
| Abnormal gel migration and separation | Incorrect buffer type | • Make sure the gel is prepared in the running buffer (1x TAE buffer >) (Genaxxon product id: M3085) |
| Sample containing unusual sequences or conformations | • Using more than recommended intercalating dye may change the conformation of DNA plasmid leading to its different migrant speed • AT-rich DNA may migrate more slowly in high-resolution electrophoresis. • Methylated, or labeled DNA with biotin or large fluorescent molecules may migrate slower than unmodified DNA of the same base pair length. | |
| Proteins bound to nucleic acids | • Add SDS > (Genaxxon product id: D2023) in the loading dye and heat the sample, to avoid any interaction between proteins and nucleic acids, for example, after restriction digestion and ligation. | |
| Incorrect quantitation result | Incorrect ladder | • Make sure to use a ladder specifically designed for gel quantitation as it contains fragments of known quantity for each band. • Try our 50bp ladder > (Genaxxon product id: M3072) or 100bp ladder ready-to-use > for shorter fragments and 1Kb ladder ready-to-use > (Genaxxon product id: M3084) for larger fragments. • Other ladders can be found on our webpage > |
| Different loading dyes for ladder and for samples | • Use same loading dye throughout to maintain uniformity and have same migration speed for the ladder as well as samples. | |
| Improper intensity measurement | • For achieving more precise intensity measurements, the measured value of the band of interest needs to be normalized to that of the background intensity |
