August 21, 2022

Agarose Substitution for Electrophoresis - Week 2

After planning this study for oh so long, I finally got to begin actual experimentation! The schedule I planned for this week was to make the control gels and prepare a DNA sample for amplification. Since something more eventful than paper writing was done this week (despite me considering paperwork quite eventful), today's post will be a dive into the specifics of what tasks I did.

Gel Creation: Controls

My first order of business: figure out what the gels made in the previous paper were like. Luckily, the standardization of documentation and academic literature makes the process for me to confirm their results a breeze. I simply replicate their study and make the same corn starch gel the researchers made. The reason why this is especially important is that I need to confirm whether or not their paper was faulty. It's totally possible that their gel could've simply worked due to an error (considering there is only one trial conducted in the actual study). The other reason why replicating their study is important is that it helps me figure out the nuances in conducting a similar experiment. For instance, during the process of replicating the corn starch gel, I figured out the optimal time necessary to allow the starch to settle before incubating and the optimal rate at which to pour the modified starch into the boiling TAE mixture. With this information, I can conduct the actual trial using the tapioca starch more efficiently.

So for this week, the two gels I made were an agarose gel and a corn starch gel. I made the gels according to the protocol presented in the paper, except the gels were cut in half and put together onto the same gel bed. This allows me to run both of the gels in the same environment, helping to eliminate potential errors from running them in two separate boxes. This "chimera gel" has yet to be run, however, the gel is prepared once the amplified samples are ready.

A photo of the "chimera gel" (corn starch on the left, agarose on the right); notice the difference in clarity between the two gels

As said in a past post, no experiment should be done without reason. So for those of you who read my posts and pay attention, you may realize that I might be being hypocritical and amplifying DNA in a study that doesn't involve PCR.  Well luckily for my readers, I am still true to my word! The point of gel electrophoresis is to be able to separate out DNA by the number of base pairs (referred to as bp) in each segment. However, it's important that the gel can actually separate out DNA accurately and not just migrate in general. For this reason, I took a sample of mtDNA (which was humbly donated by Jerry; your sample will forever be remembered in my heart) and amplified it. The specific segment being amplified with my primer set is ~250bp. Using this, alongside a DNA ladder, I can determine the accuracy of the migration and ensure that the gel substitute can actually be used effectively.

Closing Remarks

This week will mark my first set of results for the control. The further I progress in my research, the more hopeful I am about the potential that tapioca starch will be a viable option. Just looking at the gel for corn starch, you can see how opaque it becomes. I genuinely believe the issue with no bands appearing is due to this opaqueness and not an issue with the rxn between the DNA and the stain not occurring. However, the study is still in its early stages. My hypothesis can easily be proven wrong, but that is to be seen!