Sp'23: Week 12 (4/10/23 - 4/14/23): An Attempt at Transformation

This week, I can happily say that I was able to attempt to perform transformation once more, although the results are still pending. 

I started this week by checking out the concentration of last week's plasmid extractions by doing nanodrop readings on my four test tubes: "4.7A," "4.7B," "4.7C," and "4.7D," all of which had been inoculated with E coli with the plasma pRad1 in LB broth. Unfortunately, the dsDNA readings done with the nanodrop, which was blanked with LB, were much lower than expected, with the following concentrations (in ng/uL) listed respectfully: 45.4, 67.3, 48.9, and 91.6. 

Now, the ideal number that I am attempting to work toward in order to move forward with transformation would be a dsDNA reading of around or at least 200 ng/mL. What I did end up trying was seeing if I might be able to pack any of the plasms together to increase the concentration of the plasmids I was working with. I did attempt to combine plasmid from tubes 4.7A and 4.7C, who's readings were 45.4 ng/uL and 48.9 ng/uL respectfully. However, after all was said and done, I was only able to bring the concentration up to 56.5 ng/uL. I did try once more, and that only increased it by about 2 nanograms to a concentration of 58.8 ng/uL. After asking about the idea of plasma packing with one of the lab mentors, he ended up letting me know that we actually don't have the machinery that provides a high or fast enough spin to really pack the plasmid in the way that I would want to (but it was worth a try!). 

Because of this, I ended up moving on and went for a transformation using the fourth tube (tube 4.7D), which had the highest concentration of almost 100 ng/uL. To transform, Jonathan allowed me to use his competent D. Aquaticus cells. With the amount of plasma that I had extracted, I was able to attempt a total of two transformations and place them overnight into a 30° Celsius shaking incubator.

The next day, a fellow teammate was able to run Day 2 of transformation for me as transformation is a two-day process that runs back to back (and I was unable to come in to complete Day 2). 

When I checked for a transformation on the plates on Friday, which was the day after day two of transformation was performed by my fellow lab mate, and unfortunately did not see any growth on the plates: 



Because of this, I placed them back into the incubator to see if they would grow more over the weekend. With this, the initial plasmid readings taken two days before were quite low as well, so I ended up re-inoculating my E coli with the pRad1 plasmid into two new tubes of LB + Ampicillin [50 ug/mL, which is E. coli's mic concentration) for growth over the weekend (tube names "4.14A" and "4.14B").

We will see what happens with the growth come next week!

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