I was in dire need of a break after the busy spring path which I had plotted for myself. After months spent tutoring three classes, mentoring the college prep class at Carl Hayden High School, interning in the lab, and working my job at the Arizona Center for Nature Conservation (The Phoenix Zoo), I decided to work full-time and loaf around while I waited for the enrollment email from Phoenix College. I got to spend a lot of time with my crazy dogs, Mr. Bentley (my pit bull) and Samuel Marshall the Third (my labradoodle). I also squeezed in a bunch of relaxation time, as well. But now it is time to get back to work. I am looking forward to it!
I have decided to move my research project into a slightly different direction. To date, I have been working solely with gram-negative bacterial species. Last semester, I tested multiple techniques for extracting DNA from gram negatives and tweaked every aspect of my project so that each procedure would maximize results from that particular type of bacteria. Even the primers which I developed for use during PCR amplification were based solely on the available 16s ribosomal gene maps of gram negative strains.
This initial focus, of course, was due to the fact that gram negative cells are easier to lyse because of their cell wall structure. Gram negatives, despite having a double-membrane, contain only a thin layer of peptidoglycan between the two membranes. This small amount of peptidoglycan can make cell lysis and DNA extraction easier; that ease of cell disruption can also lead to sheared or destroyed DNA when an extraction is conducted using a lysing additive that is too harsh for this type of cell wall. As a result, I had quite a bit of testing to do of various protocols before I settled on the simple thermal protocol using an SDS/TBS mixture which I described in last semester's research paper.
As I have at this point repeatedly extracted and amplified DNA from gram negatives, I am extending the project to include gram positives. Gram positive cells essentially contain a single cell membrane surrounded by a thick layer of peptidoglycan. This thicker layer (than that which is contained in gram negatives) can be difficult to lyse during an extraction. I have tested my gram negative extraction protocol against several gram positive strains; it was unsuccessful at producing DNA. Therefore, the first step of this summer's project will be to develop a simple extraction protocol for gram positives.
From there, I will test my gram negative nucleotide primers against the gram positive bacterial DNA to determine their viability for use with both types of bacteria. If they are proven to be unsuccessful at amplification, I will return to analysis of the 16s gene maps of all of my target species in an attempt to design new, universal PCR primers.
That is the primary focus of my summer project. I will keep you updated as to my progress.
Until next week, when I can begin posting data and photographs relevant to my project, please enjoy this pic of my dog pack. :)
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