RNA cleanup & qPCR

Introduction 
    The main focus of this week was completing set-up for quantitative polymerase chain reaction, or qPCR. This was a shortened week with the Monday holiday, so a large portion of the week was dedicated to setting up and then running qPCR, with a bit of time being set aside to work on tests for the Sonorensis project, specifically sugar fermentation and more experimentation on TGY vs. R2A. At the beginning of the week we had a discussion with Chad about microscopy, though for those on the AMC project it was shorter than for those on sonorensis.
Methods
   


Shown above are the calculations done for qPCR setup
For each of the sugar fermentation tests, they were inoculated via our usual method:
  • inoculation loop is heated
  • loop is briefly cooled, then used to collect bacteria to be added to other medium (sugar fermentation tube in this case)
  • collected bacteria is added to new media (sugar fermentation tube) then allowed to grow
Tubes were inoculated with deinococcus sonorensis (test), micrococcus luteus (control, negative), and staphylococcus epidermis (control, positive).
Results
Based on our qPCR run, it was decided that our results, though positive, were too small to really be considered significant. UV test will be run again but at higher levels to further test e. coli's ability to recover from ultraviolet radiation. Sugar fermentation tests were left over the weekend to grow.
Discussion
    While this week did largely focus on qPCR, we have not had the discussion about how that works yet, but it is scheduled for next week. Instead, this week for the sonorensis project there was a discussion on fluorescent microscopy and how it works, as well as the importance of picking the right dyes. Depending on what we want to/expect to see with sonorensis will determine what color dyes we should use. Dyes tend to highlight certain structures so if there is a specific structure we are expecting to see within sonorensis then we should use the correspoding dye to locate the structure, if it does exist within the cells. 
Conclusion
    For next week, most of the time will likely be spent on sonorensis and characterization tests. There wil also definitely be a day dedicated to learning about qPCR and how it works, as well as how to interpret results. Because our results from qPCR on e. coli were decided to be less than stellar, we will also likely work on another round of RNA isolation followed by qPCR.

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