This week, I resumed the experimentation phase of my project after a brief hiatus caused by the thermocycler being unavailable. Without that essential piece of equipment, I have been unable to conduct any experiments, as the particular phase I am at within my project requires regular polymerase-chain reactions (PCRs) to test my hypothesis regarding universal primers. I did tinker around with some DNA extractions in the interim, as I am using some new base organisms for the project.
I experienced some minor hiccups in the extraction process of the new species, which after some review I have decided is a result of my switch to a new growth medium for my bacteria. To date, I had been using luria broth for cultures; with this new round of extractions, I switched to TSB. This richer medium is causing me to experience significant increases in culture growth during my standard 24-hour incubation period, which is resulting in larger pellet sizes upon centrifugation. These larger pellets were presenting some difficulty during the re-suspension step, and the enhanced vortexing required to properly mix the solution potentially resulted in an unusable extraction sample. After precipitation of the DNA was attempted using isopropanol, I had samples that were viscous and would not pellet. This caused me to not be able to retrieve a clean sample from the supernatant, and the extractions had to be discarded.
I repeated the process and adjusted the centrifuging times to account for the growth increase, which produced a less-dense sample pellet that was more easily re-suspended in solution. As a result, I was able to cleanly precipitate and isolate my DNA samples from the added bacterial species.
I then was able to proceed to the next phase of my project, which is the testing of nucleotide primers that I designed using genetic maps of the 16s ribosomal sequences of three of my target organisms. As you can imagine, I was extremely excited to finally get to this phase of the project.
I will post the results of this exciting new experiment on my very next blog, as I am still reviewing the data generated by the reaction. Until then, have a most excellent week!
Please enjoy the following abstract about the importance of 16s ribosomal gene sequencing; please download the full paper if you are so inclined:
http://www.ncbi.nlm.nih.gov/pubmed/18828852
And this:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523561/
Plus, enjoy this representation of an enzyme used for PCRs:
Source: www.biochem.arizona.edu; DNA taq polymerase enzyme structure
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