This week, I spent a great deal of time refining my extraction protocol to account for the increased growth encountered after I switched mediums from luria broth to TSB. I adjusted the initial sample size used to create a pellet for the extraction from 4.5 mL to 0.5 mL, and and reduced the amount of time that I centrifuged and vortexed each sample during the protocol application by 50% or more. This resulted in viable DNA samples for five of my eight bacterial species; the remaining three did not produce DNA. Because of the late date of the semester and the fact that I need additional data before I can write my research paper, I have decided to switch back to the growth medium I was previously using successfully for all eight species. This will allow me to get the data I need without losing any more time tinkering around with a basic procedure.
I also was able to spend some time running a PCR on one of the primer sets that I previously designed and have been excitedly waiting to test. Unfortunately, and to my profound disappointment, I have decided that the data generated from that experiment is unusable due to an error in the protocol used to provide the DNA sample used in the reaction.
I am going to re-test the reaction using another set of DNA samples that have already been evaluated for DNA using an electrophoresis gel. I have been storing the samples on ice for just such an occasion as this. The samples have been repeatedly subjected to gels, and have showed clear banding every time for all eight target species.
Sometime next week, I will set up a reaction using those samples and the primers I have designed. In the interim, please have an excellent weekend.
Please enjoy this photo of Proteus mirabilis, one of the species I am using. This little guy is commonly found in the human gastrointestinal tract, soil, and water. It can cause a number of infections in humans, and has been linked to the formation of kidney stones. (Davis & Zuber)
Link for more information from Davis & Zuber: http://www.clinicianreviews.com/specialty-focus/nephrology/article/iproteus-mirabilisi-isolating-a-cause-of-kidney-stones.html
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