Week 12-Semester 3. Finally, PCR results.

Hello! Welcome back to my blog.

This week, I was able to get lab time on the thermocycler, and thus began the final phase of my project. This last portion involves testing the nucleotide primers that I have designed for universality against the nine bacterial species selected for this project. I have already successfully tested a number of Universal Rice Primers against my extraction samples, so all that remains is to test these new primers in various combinations and at a number of different annealing temperatures and then analyze the data that provides.

Theoretically, the series of primers I am testing should successfully amplify DNA in three of my samples because the nucleotide sequences they contain were developed using the genetic sequences of those three bacteria. Therefore, I ran the first PCR of this project phase on the species S. enterica, S. sonnei, and K. oxytoca. I used an annealing temperature of 64°C during the reaction, which was squarely between my optimal primer temperatures of 62.4°C and 68.7°C. The results? DNA! However, the clarity of banding was extremely poor for S. sonnei and K. oxytoca. This may have been an indication of a number of things, including the amount of DNA present in my original extraction sample, the annealing temperature used, or the primers themselves.

In order to rule out/identify my sample itself as the potential culprit, I ran another PCR reaction using DNA samples, from a different extraction date, that were previously amplified using Universal Rice Primers. All controls in the reaction were an exact duplicate of the first PCR I ran this week.

The results? DNA, clearly visible for all three bacterial species, in my post-PCR gel.

This is a success for me on two fronts. One, it proved my hypothesis that my sample was responsible for the results of experiment #1. Two, it showed that the primers I designed work. This makes me quite happy, for it is an affirmation that my rudimentary understanding of genetics is improving.

Next week, due to these promising results, I will expand my PCR reactions to include the remaining organisms in this study. Until then, enjoy yourselves..and enjoy the following information about Neanderthal DNA that was recently extracted from an Italian specimen.

http://www.livescience.com/50458-oldest-neanderthal-dna-found.html

The above links to background information about Altamura man, a specimen of Homo neanderthalensis found in a cave in Italy approximately 150,000 years after his death.

This image shows the stalactites covering the specimen. Source: earth66.com