6.2 Electrophoresis

Electrophoresis is the process of moving charged particles (in our case DNA) in a fluid or gel under the influence of an electric field. To analyze a target gene with electrophoresis, PCR is preformed first to amplify the DNA concentration of the target gene. The end goal of electrophoresis is to effectively estimate the number of base pairs in the DNA samples that are being tested in order to identify the alleles that are present.

Generally, we use the QIAxcel for capillary electrophoresis, which is much faster than gel electrophoresis. However, we also have the reagents and equipment to run a classic gel electrophoresis experiment using ethidium bromide. Check out the video below to get a better understanding of how to run a manual gel.

6.2.1 Analyzing Gels

Analyzing gels can help give you different pieces of information. If your PCR experiment has been implemented with newly designed primers, then a gel can help validate that your PCR is working correctly. Running PCR experiments with positive and negative DNA controls is a good way to confirm that new primers are working. Strange PCR results generated by new primers may indicate that your experiment needs to be manipulated. This could mean that you need to redisign your primers, new reagents need to be made, or you need to alter your PCR conditions.

After you have designed and validated your PCR experiment with positive and negative controls, you are then able to run your PCR experiment on untested DNA. Measuring the bands of the PCR experiment will tell you if the organism of interest is a heterozygous dominant, heterozygous recessive, or homozygous. If different alleles of a gene are simply a SNP, gels aren’t usually precise enough to measure this difference. In this case a restriction enzyme could be used to break apart the gene at the location of the SNP. That way you can determine which version of the allele is present based on the absene or appearance of multiple DNA bands.