Week 7 (10/17/22 – 10/21/22): Plasmids and LB and Nanodrops -- Oh My!

This week was about solidifying my understanding of why certain protocols were in place for different processes we worked on (and as always, lots of learning opportunities). This week, our focus was to be on extracting plasmid, running a gel, and beginning transformation, but alas, the data we obtained did not allow for us to move forward with this task quite yet. 

After running three different plasmid extractions from E. coli (one per each of the plasmids we have been focusing on: pRad1, pRadZ1, and pRadZ2), wherein all steps were meticulously followed, we noticed that the data obtained from the nano drop (as in the dsDNA reading that provided a concentration of plasmid) was extremely low: 

Because at least 1 microgram (equivalent to 1000 nanograms) is needed to run the experiment, you'd use the calculation of dividing 1000 by the concentration (since the concentration, as seen above on the nanodrop reading, is measured in ng/uL) to figure out the volume of plasmid that would be needed both to run a gel and for transformation itself. Now, for them to be solid numbers to work with, each of the concentrations for the dsDNA readings should be around at least 70 or 80 ng/uL, but with readings so low, it was obvious that we did not have enough plasmid to work with, which likely means that most of our bacteria that we had extracted from were dead (I then learned that it is important to run plasmid extractions ideally the next day after inoculation, as long as the numbers are decent of course). 

With this, we ended up needing to switch gears and decided instead to create LB Agar plates with ampicillin so that we can inoculate / plate fresh E. coli from freezeback that has the correct pRadZ1 plasmid. We also created 2x LB broth so that when we grow our E coli on our plates, we can inoculate from there into this more concentrated LB to help with the bacterial growth (2x LB broth has double the concentration of tryptone, yeast extract, and salt)-- See below: 

By doing this, not only will we likely have solid growth in the end in the 2x LB broth tubes that we will be inoculating into, but we can then have our own plates of E coli with the pRad1 plasmid (this way we don't have to keep asking our mentor to borrow his :)) and can create our own freeze back. 

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