Week 13-Semester3. A Breakthrough!

Hello! Welcome back to my blog! Good news to report in this edition.

Last week, I had some initial success amplifying DNA from S. enterica, S. sonnei, and K. oxytoca using the first set of primers I had previously designed. As you may recall, I conducted a PCR reaction that showed positive results (for DNA amplification) and then repeated the experiment using a different set of DNA samples (in order to repeat the results and establish a pattern of amplification). Based on the data from those PCRs, this week I expanded my experiment to include the other organisms in my study. Using the same PCR protocol and primer set, I set out to see if amplification of the other species was possible.

The result? Of the nine species identified for this project, a total of five amplified during the third PCR. I considered this to be promising, as the fact that there was amplification indicated a number of things:

• The technique used to identify nucleotide sequences in the gene for potential amplification worked.
•  The annealing temperature parameters selected for the tested primers worked. 
• Given that amplification was demonstrated to be possible, testing of the other primer sets designed for this study could proceed.

This in itself was promising news. However, I still needed more data for my research paper, so I decided to expand my testing to include the next set of primers in the queue.

Therefore, I immediately set about running another PCR. I duplicated all controls from the first three reactions, exactly; even the annealing temperature remained the same, due to a similarity in optimal temperature ranges between the two sets of primers.

The results of this new test? Success! All nine of my organisms showed DNA banding in the post-PCR gels. Meaning that for the first time and after a year of testing, I was able to amplify DNA from all of my target organisms using one primer set of my own design. This, of course, is data that made me quite happy.

Also of course, this data is only preliminary; I will need to re-create the results a couple of more times, using a molecular marker, before I can be certain of the results' validity.

Looks like I know what I will be doing for the rest of the semester!

Until next week, cheers.

Paul C.

PS: Here's a link to a video about potential cloning using Mammuthus primigenius DNA.  http://www.sciencedaily.com/videos/5afc2e6cc20523ee3fc44352717bb039.htm

Photo credit: a-z-animals.com. The wooly mammoth in the collection of The Royal BC Museum, Vancouver, BC.

PPS: For more details about the work I have done this semester, including protocols, supporting data, and gel images, my research paper will be available for review via a link on this blog at the end of this semester.