Sp'23: Week 14 (4/24/23 - 4/28/23): The Week of Kit Comparisons

Quite a bit was accomplished this week in preparation for another soon-to-be-attempted transformation of Deinococcus aquaticus using the plasmid pRad1 extracted from E. coli. To begin, nanodrop readings were done on both tubes (tube "4.21C" and "4.21D") that had been inoculated last week with E. coli + the pRad1 plasmid into LB + Ampicillin [mic concentration of 50 ug/mL]. Now, usually E. coli when placed into the shaking incubator at a lower growth temperature of 32 degrees Celsius will grow over the weekend and can be measured a few days later to show growth. However, the OD600 readings showed negative readings, which indicated no growth, and verified that E. coli likely never grew in last week's LB tubes (tubes "4.14A" and "4.14B") either. The respective readings of tubes "4.21C" and "4.21D" were as such: 


These negative readings, indicating no growth, unfortunately meant that I would need to re-inoculate E.coli with pRad1 from our freezeback stock. Either way, I knew that to have a freshly grown batch of E. coli with pRad1, it would be best to have freshly-made LB plates (both with and without Ampicillin [mic concentration of 50 ug/mL]), so I went ahead and made two sets of 125 mL LB broth + Agar (one of which would eventually have Ampicillin added to it before pouring the plates). 

After these two sets of LB + Agar were autoclaved, I used them to create two sets of 5 plates each. Five of these plates (plates 4.24B1 - 4.24B5) would purely be made of LB broth + Agar: 

While the other five plates (plates 4.24B6 - 4.24B10) would each ultimately be made of LB broth + Agar with a calculated Ampicillin concentration of 50 ug/mL (which is the mic concentration for E. coli): 

I did go ahead and allow all ten plates to dry as much as I could that day, but only placed the + Ampicillin plates into the fridge (onto two of which with +Ampicillin, plates "4.24B6" and "4.24B7," my teammate Alex was able to inoculate E. coli + pRad1 from freezeback the next day). The -Ampicillin plates were allowed to dry overnight and were placed into the fridge the very next day. Now luckily, plates "4.24B6" and "4.24B7" did show plenty of growth overnight. Because of this, I was able to inoculate both plates straight away into new sets of centrifuge tubes that held LB + Ampicillin [E. coli's mic concentration of 50 ug/mL]. E. coli with pRad1 from tube "4.24B6" was inoculated into tube "4.27A," while E. coli with pRad1 from tube "4.24B7" was inoculated into tube "4.27B." Both tubes were placed into the 37 degree Celsius incubator. 

On another note, I did check plates "4.5B5" and "4.5B6" that contained the [hopefully] transformed D. aquaticus with pRad1, but unfortunately this was to no avail. There had been no growth this entire time, which meant that growth of transformed D. aquaticus was probably not going to occur on those plates: 

The next day, I had an opportunity to check the growth of E. coli with pRad1 into tubes 4.27A and 4.27B which held LB + Ampicillin [50 ug/mL]. Fortunately, where was growth (with respective OD600 readings done via nanodrop shown below): 

The plan this day was to run a plasmid extraction on each tube, and even though the growth showed an OD600 reading of less than 1 (which was less than ideal), I went forward with plasmid extractions (all the while, allowing the E. coli to continue growing in the tubes by letting them sit in the 37 degree Celsius incubator while plasmid extractions were being done). With this, I decided to test two different extraction kits to see if one might yield better results than the other. Luckily, this was the case! 

For the first plasmid extraction that I ran, I used the thermo scientific GeneJET Plasmid Miniprep Kit to run one plasmid extraction from each of tubes 4.27A and 4.27B, creating respective plasmid extraction tubes "4.28A1" and "4.28A2." These dsDNA readings were the highest that day overall at 110.5 and 153.8 ng/uL respectively: 


The second plasmid extraction I ran was with use of the ZymoPURE Plasmid Miniprep Kit [Box A] using tubes 4.27A and 4.27B once again. This kit yielded much lower nanodrop results when using the same tubes 4.27A and 4.27B (dsDNA readings of 66.5 ng/uL for tube "4.28B1" and 74.9 ng/uL for tube "4.28B2" respectively): 

 

The entire time that I ran each set of extractions using both kits, I was allowing the E. coli + pRad1 in tubes 4.27A and 4.27B to sit in the 37 degree Celsius incubator when they were not in use (which was most of the time while each plasmid extraction was taking place). After I had run both sets of plasmid extractions, I checked the growth of tubes 4.27A and 4.27B, and with Dr. Tuohy's help, determined that E. coli's growth had approximately doubled within the two hours or so that they were sitting in the incubator, as they were likely in their exponential growth phase (an ideal phase for plasmid extraction, or so I've read). The growth of each tube had increased in their A600 readings from 0.86 to 1.04 for tube 4.27A and from 0.81 to 1.06 for tube 4.27B: 

Because of this, I performed another set of plasmid extractions, this time doing 6 at a time (three per tube). In doing this, I used tube "4.27A" to perform three pRad1 extractions into three new microcentrifuge tubes ("4.28C1A," "4.28C1B," and "4.28C1C"), while using "4.27B" to perform three pRad1 extractions into another three new microcentrifuge tubes ("4.28C2A," "4.28C2B," and "4.28C2C"). The extractions into the six microcentrifuge tubes "4.28C1A" - "4.28C2C" resulted in the following plasmid yields and dsDNA readings with the nanodrop: 84.5, 91.5, 95.5, 87.7, 125.3, and 99.4 ng/uL. 

I used the thermo scientific GeneJet Plasmid Miniprep Kit once more, as this was the kit that provided higher plasmid extraction yield upon my own comparison. The numbers were still higher than the plasmid extraction yields from the ZymoPURE Plasmid Miniprep Kit [Box A], but were lower than when extraction was run with the thermo scientific kit the first time (even though E. coli's growth was higher, which would assumedly yield an even higher plasmid quantity). I assume this is because as more extractions are done at a time, the timing for each step of the extraction is less precise, which makes sense as to why the yield might have been lower than when using the same kit (with an even higher E. coli growth) just a few hours before. 

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