S-STEM Scholar Marisa Ostos Blog

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...

This week was more focused on planning, but I was able to perform a few tasks to continue forward with my project.  First, as it is good to have fresh LB and TGY to pull from, I went ahead and created two new microcentrifuge tubes (1 mL each) of TGY and LB broth to pull from for nanodrop readings when needed, with the following tube names:  "TGY 4-21-23 M.O. 4.21A "  "LB 4-21-23 M.O. 4.21B "  With this, I wanted to check the growth of the two inoculations I did the Friday before. Each of these tubes held LB + Ampicillin [with an E. coli mic concentration of 50 ug/mL] and were inoculated with E. coli that held the pRad1 plasmid. Unfortunately, the OD600 readings were extremely low (negative numbers), which indicated either no growth or death of the E. coli that may have grown (as they had been in the 37 degree Celsius incubator for almost a week). The OD600 values were as such:  Now, because the OD600 readings showed that there were likely...

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 pl...

After last week's unsuccessful attempts at plasmid extractions, it was back to the drawing board (aka time for fresh inoculations). First, I decided that it would be helpful to have my own fresh batch of Deinococcus aquaticus  to pull from, so I took from Jonathan's plate (plate "2.10JH") of D. aquaticus  and inoculated onto one of my own plates with TGY + Agar (plate "3.24C6" that had been made less than two weeks ago). I also inoculated from is "2.10JH" plate into four different sets of 4mL TGY 3:3:1 polystyrene tubes (labeled "4.5A1," "4.5A2," "4.5A3," and "4.5A4" respectively). The plate and all four tubes were then placed into the 28 degree Celsius incubator to allow them time to grow:  I also went ahead and made 6 TGY 3:3:1 Agar plates (named "4.5B1" - "4.5B6") with Chloramphenicol (+Cm) [3 ug/mL], which is assumed for now to be the mic concentration of the antibiotic Chl...