Performing PCR, gel electrophoresis, and PCR clean up

5 March 2022
Brooke Andel and Spencer Harstad

Introduction:

The Brain and Behavior research stream under Dr. Rachel Cohen focuses on the specific green anole lizards that live in the southeastern part of the United States. These lizards are unique in that they have specific seasons for breeding. Our focus is trying to determine how certain genes that are found in the hypothalamus of the brain might affect the changes that these lizards undergo physically and behaviorally. We are attempting to do this by determining how the genes are expressed throughout the breeding compared to non breeding seasons. To begin our research, we each got to select a specific gene from a pool that was provided and known to potentially influence these changes we see in the lizards. The two genes we selected are cold-inducible RNA binding protein (CIRBP) and hormonally upregulated neu-associated kinase (HUNK).

Figure 1: Spencer and Brooke placing PCR tubes into the thermal cycler.

Research:

Over the last few weeks, we have been creating polymerase chain reactions (PCR) for each of our designed primer sets and running them through gel electrophoresis. PCR is a method to amplify and create a significant number of copies of a specific region of DNA. We created a PCR master mix for each primer set we ordered. We had three primer sets for our CIRBP gene and two for HUNK. Within each master mix contained the Quick-load TAQ 2X (DNA polymerase), PCR water, and the forward and reverse primers that were specific to each primer set. After the five master mixes were created, we split them into two tubes. This allowed us to add cDNA to one tube and water to the other. We did this for each master mix, so we were able to have a positive control with the cDNA and a negative with the water. Single stranded RNA is converted by reverse transcriptase into complementary DNA (cDNA). We use cDNA to allow for amplification when performing PCR. The cDNA we used comes from the lizard hypothalamus that is provided to us. The water we use is not just ordinary water, it is distilled. We use this distilled water because it essentially is inert, which means there is nothing but oxygen and hydrogen in the water. This is crucial because we do not want any other matter or minerals in the water that could affect the interactions between all the components of the PCR. When we eventually run the PCR through a gel, we should see clear amplification of the samples that contain the cDNA and no amplification in the negative controls that contain the PCR water if everything was performed and measured correctly. However, before we can see amplification, we must allow for the amplification of the PCR by allowing it to process through the thermal cycler. Once the thermal cycler is done, we are now ready to create our gel and load our PCR samples into it.

Figure 2: Brooke pipetting a PCR sample into a well on the gel.

To begin by making a gel we combined agarose and 1xTAE buffer solution and heat it up until all the agarose has been dissolved. Then we allow for the gel to cool, and then add in the extremely small amount of bullseye DNA dye. This dye is what allows us to visualize the DNA fragments as bands in the gel according to their size. Once the gel is poured into the frame and solidified, we box. This box contains electrodes on it, and it is crucial to remember to then move it into the gel place the gel, so the wells are closer to the negative electrodes while loading the gel with the PCR samples. This is because since DNA fragments are negatively changed this will cause the DNA fragments to travel towards the positive electrode side. The DNA fragments after 30 minutes will not all have traveled as far and this is because smaller DNA pieces can pass through the gel easier, they will run farther. After the gel run is complete, we take it to the imaging machine. This machine allows us to see our DNA fragments and analyze what we see. Each time we image our gel, we are hoping to see strong bands in our wells that contain the cDNA and no bands in the wells with our negative control of the distilled water. So far, though all the gels we have run, we have seen contaminations at least once for each primer set. However, because we had no contaminants that we could clearly see in our most recent gel, we are now ready to move on to perform a PCR clean up. A PCR clean-up is performed to isolate the DNA from all enzymes, primers, and other components, so they will not interfere with our results. The results we are wanting to gain from performing a PCR clean up, is the DNA sequencing of our gene. After we perform the PCR clean up and isolate the DNA, we will then proceed to send our solution containing our DNA to get sequenced.

Reflection:

The time we have spent in the lab over the beginning of this semester has been a learning experience that has shown us a lot. For the most part, we have been successful with the results we have gotten and are proceeding to move forward. However, we have experienced minor setbacks while troubleshooting with PCR and gel electrophoresis. The issue we have run into multiple times now are pipetting errors or contamination that is most likely due to a pipetting error. As we have noticed this becoming a trend, we began to take extra precaution and be more attentive while pipetting. We are excited to continue our work and to use our skills in the lab as the semester continues.

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