After making mistakes last Wednesday with my plasmid extraction, I did go ahead and do nanodrop readings of my two sets of 2 tubes each (totaling four tubes). Here are the results:
(Figure A) The nanodrop (dsDNA) readings shown above represent each of the four microcentrifuge tubes shown below. In order, row 1 = "2.3A 1 of 2," "row 2 = "2.3A 2 of 2," row 3 = "2.3B 1 of 2," & row 4 = "2.3B 2 of 2." The tubes that were used to create these nanodrop readings are seen below:
(Figure B) These are the four tubes that were used to run the nanodrop readings seen, which correlate with the nanodrop readings.
Now, because I had forgotten to allow the elution buffer to sit for 2 minutes at room temperature before centrifuging, I did a "mini experiment" and re-eluted one of my four tubes (Tube 2.3B, which I felt was safest to experiment with as the nanodrop reading showed this tube as having the lowest concentration, and because I figured that our plasmid might be contaminated). I went ahead and placed the 25uL from Tube 2.3B into a brand new column matrix, then re-eluted it with 25 uL of the "ZymoPURE Elution Buffer," this time allowing it to sit for 2 minutes at room temperature before microcentrifuging for 1 minute. For this tube, I ended up doing 2 nanodrop readings: Once right before I added the elution buffer / incubated for 2 minutes at room temperature, and once right after I ran the elution buffer / 2-minute room-temperature incubation process. Here are the results:
(Figure C) This shows my nanodrop readings from Tube "2.3B 2 of 2" before and after my "mini experiment." Row 1 = Tube 2.3B 2 of 2 (before experimenting,) & Row 2 = Tube 2.3B 2 of 2 (after experimenting).
(Figure D) The above shows tube "2.3A 2 of 2" used for my mini experiment, including the bottle of elution buffer used to elute and blank the nanodrop, and the new [unlabeled] microcentrifuge that contained the "experimentee" ("Tube 2.3B 2 of 2" after having eluted with 25uL elution buffer and incubated at room temperature for 2 minutes before centrifuging for 1 minute).
I wasn't sure what to expect, but my dsDNA nanodrop readings dropped quite drastically to 5.5 ng/uL. So, because I had three microcentrifuge tubes left, each of which had a mistake occur with it in some way during the extraction process, I decided to redo my plasmid extraction on the [likely contaminated] tubes. I grabbed tubes 2.1A & 2.1B, neither of which had Ampicillin added to them, and used the Zippy Kit A to run extraction again. Although I should've checked the growth of these two tubes (each of which held the [likely contaminated] E. coli with pRad1) and run gram stains first, I extracted and took nanodrop readings after extracting:
(Figure E) The nanodrop readings from plasmid extractions of Tubes 2.1A & 2.1B seen below, in order.
(Figures F-H) The above includes the tubes I used to run the plasmid extraction (Figure F), the "Zippy" Kit (box A) that was used for extraction (Figure G), & the plasmid extractions placed into microcentrifuge tubes "2.8A" & 2.8B" (from tube 2.1A & 2.1B respectively).
The extraction readings were quite high, which was great as they were the highest dsDNA nanodrop extraction concentrations seen thus far (195.5 ng/uL from tube 2.1A & 184.9 ng/uL from tube 2.1B). Although I still suspected contamination, at the suggestion of my mentor to see what might happen, I went ahead and did the XbaI digest, providing two PCR tubes'-worth of digested pRad1, each holding 50uL of digested plasmid.
(Figure I) The above shows the PCR Thermo Cycler used, along with the settings required, for XbaI plasmid digestion (the thermocycler being used to hold run the plasmid at different temperatures for lysis), this way I would have an open plasmid with which to run a gel.
On Friday, a 1% gel was created for gel electrophoresis to see if I had viable pRAd1 plasmid that could be worked with for transformation. After having created and run the gel, it was observed that the plasmid was, indeed, contaminated:
(Figure J) This shows the result of the 1% gel run, with the left-most column being the 1kb ladder with which to compare DNA size. The third column showed the digested plasmid with three different bands, and the fifth column showed the undigested plasmid.
Finally, because this gel showed that we did not have clean extracted pRad1 plasmid with which to work, I re-inoculated from plate "2.1_" (which had E. coli + pRad1 without Ampicillin, thus no extra stressor on it, which isn't the ideal choice for extraction) into two different centrifuge tubes "2.10A" & "2.10B," each of which held 15mL LB broth (which had been created by me on 1/25/23), an Ampicillin concentration of 50 ug/mL, and inoculated E. coli [ideally with pRad1] from plate "2.1_." These two tubes were placed into the incubator at approximately 26.5 degrees Celsius over the weekend.
(Figure K) Shown above are the two tubes (Tube "2.10A" & "2.10B" that held the inoculated E. coli with pRad 1 in 15mL LB broth + Ampicillin [50 ug/mL] along with the plate "2.1_" that the tubes were inoculated from.
Next week will be focused on re-extracting plasmid from these two tubes of E. coli along with (with the help of my lab mentor) transformation of Deinococcus Aquaticus (assuming the gel shows clean pRad1 that can be used after extraction).
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