Week 13, Semester 2-Success!

This week my DNA research project yielded new results, which upon discovery nearly had me leaping into the air from excitement. However, I refrained from doing so lest I give lie to the calm and studious façade I have cultivated this semester, and instead allowed myself only a silent "Hallelujah" and a brief victory trot through the lab to share my results with any available listeners.

A slight change in electrophoresis gel creation methodology yielded the clearest DNA banding I have yet received during this project. I have tested different gel recipes to-date, with varying results, and recently I began testing the optimal conditions for adding the DNA stain SybrGreen directly to my uncongealed gel solution before pouring and setting the gels. Previously, I simply added the DNA stain into each well as I added the DNA sample for testing; however, these gels showed limited or zero presence of DNA banding. To test the hypothesis that a direct addition would result in improved gel performance, I conducted a comparison experiment of the two gel types. I prepared four DNA samples using an identical protocol. I then created two gels from the same buffer/agarose base. I poured one gel, added DNA stain directly to the remaining mixture, then poured the second and allowed both to set. Finally, I loaded the samples. After thirty-five minutes running @200 volts, gel one showed zero DNA present, yet gel two showed clear banding. While this result was expected, it was exciting nonetheless.

Gel protocol:

The gels utilized for this test were created using a 0.9 g agarose/100 mL buffer mixture heated for 1:45 minutes on high in the microwave. Immediately after heating, gel #1 was poured. The remaining 75 mL mixture was allowed to cool to exactly 60°C, and then 3µl of 10,000x concentration SybrGreen was added and diffused into solution using a five-second swirling action. Gel #2 was poured, then both gels were allowed to set for 20 minutes in darkness (to prevent degradation of the SybrGreen due to UV light). The buffer mixture contained 980 mL/20mL de-ionized water/50X TAE buffer stock.

I was also able to expand the project to include additional bacterial species, which was another turn towards progress as my original hypothesis theorized the existence of universal primers that can be applied to multiple bacterial species. To-date, all experiments have utilized only E. coli. With this experiment, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus were also subjected to extraction and gel testing. All four species yielded positive results after electrophoresis, with the gram-positive S. aureus providing the most limited results. These were the results I both projected would occur and hoped for. After some additional testing to replicate this week's results with the added bacterial species, this project should now be able to move forward to the next phase, which is intended to focus on primer design.

Altogether, it was a successful week. Please enjoy this photo of my gel results until the next blog. Cheers!

Left to right: E. coli, P. aeruginosa, P. vulgaris, S. aureus

Week 12, Semester 2

This week is focused on expanding my extraction protocols to include additional bacterial species. To date, E. coli has been the sole species utilized for all extractions and polymerase chain reactions(PCRs). The additional species that have been included are : Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus. The two former were chosen due both to availability and their status as gram-negative bacteria; the protocol I am using for extractions was specifically developed for application against gram-negative species, due to the structural differences that exist between gram-negative and gram-positive species. S. aureus, though gram-positive, was selected both to test to efficacy of the gram-negative protocol against a positive species and to be used as the base species for design of a gram-positive specific protocol. It is expected that my current protocol will be ineffective at producing DNA from a gram-positive organism due to the gram-negative specificity of the cell lysing agents used in the developed protocol, thus a separate protocol will have to be designed.

Until the next blog, please enjoy the following links.

Everything you always wanted to know about unlocking and sequencing the human genome can be found at this fascinating Smithsonian website:
http://www.genome.gov/smithsonian/

Gain access to my personal favorite scientific study, the Gombe Research Project, which Dr. Jane Goodall has been conducting for more than 50 years; use Google Earth to enjoy the panoramic views of chimpanzee individuals, their family groups, and the Tanzanian park within which they reside.
http://www.google.com/maps/about/behind-the-scenes/streetview/treks/gombe-tanzania/

Photo credit: Fanni, photographed by Anup Shah for National Geographic Magazine.
Access additional photos and background information at:
http://ngm.nationalgeographic.com/ngm/0304/feature4/zoom4.html

Week 11, Semester 2-A Much-Needed Break

Hello! Last week, I was able to join our fellow scholars and faculty advisors for a welcome bit of rest and relaxation on our first field trip of the semester. We piled into the van and headed for Dreamy Draw Park and Recreation Area to hunt for an (alleged) alien crash site, do a little hiking and geo-caching, and eat lunch courtesy of Phoenix College. Who knew exercise and getting a sunburn could be so fun?

Even though the nearest Starbucks drive-through was miles away, somehow we managed to have a great time, use GPS, and learn about ephemeral desert washes before the day was over. No alien life forms were spotted, but somehow it didn't matter when I looked around at the smiling faces of this semester's team of scholars and faculty and realized once again how lucky I am to be part of this crew. It saddens me to think that another semester has almost come and gone, and before long this program, for me, will have concluded.

Next week I will pick up my research again, and begin drafting this semester's research paper. Until then, please enjoy these candid pics from our outing.

Week 10, Semester 2

Hello! And welcome back to this blog.

This week has been focused on reviewing the data collected thus far and determining the next steps of my research project. I have decided to analyze genomic sequences of the various bacteria named in my abstract for sequence correlations; this will allow me to predict a primer design that will result in DNA amplification during the polymerase-chain reaction (PCR) process. This design prediction is in line with my original project hypothesis, which is that there are universal primers for PCR usage which will successfully amplify DNA from seven common bacterial species (all species are named in my abstract; see blog Week 6, Semester 1 for details.)

After primer design is complete, the next phase of this project is to repeat the procedures used to conduct extractions on E. coli. The entire protocol testing process will not be repeated; only the protocol and precipitate solutions found to be successful at extracting E. coli will be used on the six remaining species. Then, bacterial samples from all seven species will be submitted for PCR amplification using the specific primers that were designed.

This next phase begins in earnest in Week 11. For my remaining lab time this week, I will be joining the Biology staff and other S-STEM scholars on a much-needed off-site field trip to the Dreamy Draw Park & Recreation Area. For those of you who are unable to attend, please enjoy this shot of our beautiful desert, taken from within the park.