Week 6 (10/10/22 – 10/14/22): Checks & Balances

This week served as more of a checks and balances than anything, of which I am glad there are certain protocols in place (and reasons for there being so) that Ensure we are running our experiments in the best manner possible.

Since we have not had success in running transformation three different times on plasmids that are almost 10, 000 base pairs in size, we are now focused on making sure that we can transform with a plasma that is just under 2/3 of the size of the ones we had been working with. This plasmid is called pRad1, and what's interesting about this plasmid is that the other two plasmids we had been working with which are larger are, in a sense, derivatives of this grandfather plasmid. They all have the marker which allows for resistance to ampicillin and chlorophenical, and this one can be thought of as being more of the bare bones plasmid of our other two, the "grandfather plasmid" in a way.

Now, since last week we had run three sets of inoculations to help grow E coli that had each of the three plasmids (one plasmid per E coli), this week's focus on pRad1 led to an XbaI digest (in which our circular plasmid is cut at the XbaI restriction site to make it linear instead) and an electrophoresis gel run (which helps to check the size of our plasmid and help ensure that we do in fact have the correct one). 

Seen above is the results of our electrophoresis gel run (which my lab partner was kind enough to label for ease of visual understanding). When we ran the gel, we loaded three different wells: the first one on the left represents the ladder (our measuring and comparison tool of which the highest line within that column represents the greatest size at 10,000 base pairs); the second well was our digested plasmid (the one that had been cut so that it was linear rather than circular); and for the third well that we loaded, that one showed our undigested (uncut / circular) plasmid. The second well, representing the undigested one, is the key one to focus on as this heps us figure out how large the plasmid we have actually is. Unfortunately, you can see that the undigested plasmid line is at approximately the same height as the highest line on the ladder, which means that the plasmid that we had extracted and run was actually one that was around 10,000 base pairs long. So the fortunate thing is that running the gel helps to double check whether or not we have the correct plasmid. The not-so-fortunate thing is that the pRad1 plasmid is 6,809 base pairs long, which means that the blue line representing the undigested plasmid should have not shown up near the top of the ladder, but should rather have been closer to 1/3 of the way lower than the height of the top-most line of the ladder itself. 

What did this mean? It meant that we did not have the correct plasmid. How did this happen if our tube was labeled pRad1 (which we would assume would help us know that we are taking plasmid from the correct tube which had had the correct E coli with the pRad1 plasmid inoculated)? My thoughts are that has happened for one of two reasons: the first (and highly unlikely one) is that the freeze back sample that we pulled from didn't actually have the E coli with the correct plasmid to begin with (I don't believe this is the case as I trust the sample that we pulled from,  l since this one was created by our mentor who had worked with the same plasmid before -- I'm only stating all possibilities as a just-in-case). The more likely reason is that, upon my inoculation of the E coli from my mentors freeze back sample, I misread the labels on my tubes that I was inoculating into and accidentally inoculated the pRad1 freeze back into the pRadZ1 tubes (and vice versa) -- a simple, yet crucial mistake that pushed back our timeline by an entire week. 

Although I am quite a details oriented person, the importance of double checking all labels and processes cannot be undermined. Because of this, the rest of our focus was on creating TGY agar plates (with and without chloramphenicol) to prepare for ultimate plating during the transformation process for the next week.

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