Sp'23: Week 15 (5/1/23 - 5/5/23): Strong Yields

This week, I started out by using the nanodrop to first check the growth of E. coli with pRad1 in tubes "4.27A" and "4.27B," both of which had been used for multiple extractions last week, and both of which had also been placed back into the 37 degree Celsius incubator for the weekend (which was done to see how the E. coli would grow). Overall, the nanodrop showed slightly lower OD600 readings, which means that the E. coli were out of their exponential growth phase and had growth that had likely come to its plateau (last week's final OD600 readings were at 1.04 and 1.08 for tubes "4.27A" and "4.27B" respectively). The OD600 readings for the same tubes are shown in their respective orders: 


Since the growth had not changed much, I decided that it was safe to move forward with a single plasmid extraction per tube (as I theorized that doing less extractions per tube would result in a higher pRad1 plasmid yield). I once again used the thermo scientific GeneJET Plasmid Miniprep Kit (which last time gave a higher yield of pRad1 when compared to the ZymoPURE Plasmid MiniPrep Kit [Box A]). Lo and behold -- I was correct! The resulting extraction tubes labeled "5.1A" (which had plasmid extracted from tube "4.27A") and "5.1B" (which had plasmid extracted from tube "4.27B") showed nanodrop dsDNA readings that were extremely high: 153.3 and 168.3 ng/mL respectively: 

 

What was nice was that this was some of the highest consistent plasmid yields I have obtained from doing plasmid extractions since I started with the lab almost a year ago. Because of this, I decided to create more LB + Ampicillin [at E. coli's mic concentration of 50 ug/mL], into which my teammate Alex inoculated E.coli with pRad1 from plates "4.24B6" and "4.24B7" into tubes "5.1C1" and "5.1C2."

The day after that, I used the nanodrop once more to check the growth of the E. coli with pRad1 with an OD600 reading. The OD600 results of tubes "5.1C1" and "5.2C2" were both at A600 numbers of 0.84, so I placed them back into the 37 degree Celsius incubator to allow them time to grow some more. 

After this, I ran an XbaI plasmid digest using two of the tubes that held contents (which was hopefully pRad1) of plasmid extractions from last week: tube "4.28B1" (last week's dsDNA reading was 66.5 ng/uL) and tube "4.28B2" (last week's dsDNA reading was 74.9 ng/uL). I went ahead and used the CutSmart kit to cut the circular double-stranded pRad1 plasmids at their single XbaI site, which would result in an intact and elongated / linear (rather than circular) pRad1 plasmid. 

For the digest to occur, two PCR tubes of 50 uL each of pRad1 (which had already had the XbaI restriction enzyme and buffer added) were placed into the PCR / thermal cycler machine, which had a preset setting of three different heat phases to ultimately help with plasmid digestion (Phase 1: 37 degrees Celsius for 15 minutes, Phase 2: 65 degrees Celsius for 20 minutes, and Phase 3: 4 degrees Celsius for an infinite amount of time -- which is used as the holding temperature until I am ready to take out the newly-digested plasmid). 

Once the plasmid was digested, I used a 1% gel that I created to run gel electrophoresis, which would help ensure that, with comparison to a 1kbp ladder (which measures DNA that is up to 10,000 base pairs long), I did indeed have the correct plasmid. My lab mentor, Jonathan, kindly awaited the gel results and sent them to me, which verified that I actually did extract the pRad1 plasmid. From left to right, Well #11 had the 1kbp ladder for comparison, Well #3 had a digested sample, Well #5 had an undigested sample, and Well #7 had another digested sample. 

Because of this, I felt certain that these electrophoresis results gave me the green light to run transformation once more. My teammate, Jayce, ran Day 1 of Transformation (which ultimately includes combining the pRad1 plasmid with competent Deinococcus aquaticus cells, having them undergo heat shock, then ultimately allowing the hopefully-transformed cells to grow by being given 16 hours to sit overnight in a shaking incubator at 32 degrees Celsius). With this, the following tubes were created: "5.4A1," "5.4A2," "5.4A3," "5.4B1," "5.4B2," and "5.4B3" (all six tubes contained 4mL TGY 3:3:1 and 100 uL of competent Deinococcus aquaticus from freezeback, while all except tubes "5.4A1" and "5.4B1" were given 6 uL of pRad1 from a plasmid extraction (tube "5.1B") that had a plasmid yield of 168.3 ng/uL). I ran Day 2 of transformation under the hood using asceptic technique, which included adding 100 uL from each of the 6 tubes onto different plates then placing them into the 32 degree Celsius incubator for growth over the weekend: 
  • tube "5.4A1" (-pRad1) --> plate "3.24C1" (TGY 3:3:1 -Cm) 
  • tube "5.4A2" (+pRad1) --> plate "3.24C2" (TGY 3:3:1 -Cm) 
  • tube "5.4A3" (+pRad1) --> plate "3.24C3" (TGY 3:3:1 -Cm) 
  • tube "5.4B1" (-pRad1) --> plate "3.24B1" (TGY 3:3:1 +Cm [3 ug/mL]) 
  • tube "5.4B2" (+pRad1) --> plate "3.24B2" (TGY 3:3:1 +Cm [3 ug/mL]) 
  • tube "5.4B3" (+pRad1) --> plate "3.24B3" (TGY 3:3:1 +Cm [3 ug/mL]) 

As a final task for the week, I went ahead and tried to do the first part of a plasmid extraction on tubes "5.1C1" and "5.1C2," which included packing the tubes with DNA (the idea being that I could continue with plasmid extraction next week and skip the plasmid packing, which takes a good amount of time, as it would have already been finished). These two "packed" tubes of pRad1 plasmid (labeled "5.5A1" and "5.5A2" were placed into the refrigerator for use next week): 

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