Week 5 (10/3/22 – 10/7/22): Science as a Waiting Game


First, I will start by saying science is not actually a game, per say (but it can be fun like a game!). However, it is filled with interesting turns, challenges, and acts of waiting that ultimately help to reach a final product. This week, we decided to go a different route than I had planned. The original plan was to run three sets of transformations at once to use as a type of comparative device and as a way to make sure we were running our protocols correctly. With these transformations, we would use a smaller plasmid (that had been verifiably transformed by another classmate in the past) and two larger ones that were around the same size. In this instance, if the smaller plasmid was transformed and the two larger ones weren't, then maybe we'd need to change something within the transformation protocol itself. 

Now, one interesting thing about science is that you are dealing with real-world factors, and that includes finances and availability of materials. One focus in our lab is to make sure we are using efficient methods, and since we have run transformation on larger plasmids (almost 10,000 base pairs long) three different times, with none of them working out, we decided (with Dr. Tuohy's help) to focus ensuring that we could transform on our own using the smaller plasmid. If we are able successfully transform with the smaller plasmid alone, then we'll know that we have been running correct protocols and that something might need to be tweaked in working with the larger ones. However, if we do not obtain successful transformation with the smaller plasmid (which, we absolutely know is possible as it was done using the same techniques by a classmate in the same lab), then we will need to look at our own techniques and figure out what we are doing wrong. 

Our hope and goal, however, is to make sure we can successfully transform this smaller plasmid, so we began the process that helps us get ready for transformation. After much discussion and revision of our outline, we decided to begin with an inoculation of all three E. coli (each of which have one of the three plasmids we are working with in the end). The reason we are doing this is because even though we are going to first be working on transforming D. rad with the plasmid "pRAD1," it will be good to have available our own set of E. coli that we can draw from for each of the three plasmids we will be planning on working with. With this E. coli, we will also be creating our own Freezeback, this way we have a master "stock" solution of E. coli with each of the three plasmids to draw from. We did end up having growth, albeit not the strongest numbers, and were able to run a plasmid extraction from it, whose numbers were decent enough to use for our transformation. 



Our next steps include creating plates, creating and plating the competent cells, creating Freezebacks of each of the E. coli with the three different plasmids, and ultimately transforming our cells. Hopefully we have transformation in the end!

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