PCR and Gel Electrophoresis

I used to teach a genetics lab and thought I would share what I taught my students. Before running polymerase chain reactions (or PCR), to make copies of your sample DNA, it is important to run a gel with your DNA extraction to see if the extraction worked. To run a gel, the DNA sample is loaded into wells on one side of a gel. An electrical current is used to separate out the DNA to analyze it. If your DNA extraction did not work it is a waste of money to run PCR on the sample. This gel is run in buffer TBE. TBE contains tris which raises the pH to 9, a boric acid which lowers the pH to optimizes it for the target enzymes, and EDTA which chelates metal ions and those stops DNeasy activity, which is the enzyme used for DNA extraction. The molecules in the gel separate based on charge, size, and shape. Personally, I like to use 1% agarose gel with TBE at 120 volts for 30 minutes.

gel

But what is agarose? It’s a seaweed polysaccharide that allows the gel to thicken. The more agarose you use the thicker the gel and the longer it will take your DNA sample to run through the gel. If you need to separate out really small fragments you would need a really thick gel but since we are looking to see how well the DNA extraction worked we can use a thin gel. You should also stain the DNA sample before putting the sample in the gel. If you don’t stain the sample you wouldn’t be able to see where your sample goes in the gel and run the risk if running your sample off of the gel. Gels also often have glyercide in them that binds to the DNA and makes it heavier so when you load your DNA into the well on the gel it is less likely that the DNA floats away. It another well a ladder is also typically used. In this case, we used a 1000 kb base pair ladder. A ladder is DNA fragments that are cut at known lengths. This allows it to act as a ruler for your other samples.

Continue reading