PCR and Dark Room Development (Week 4)

From the previous week, I extracted DNA from cultures grown in anaerobic conditions. These cultures were originally started from what we call a recyclostat that contains soil samples with archaea, bacteria, and fungi. On Monday of this week, I quantified the amount of DNA I extracted using a fluorospectrometer. The results were fairly low, showing that there was very little growth in the anaerobic conditions.

            Following the quantification I ran the largest PCR I have ever ran. It contained 36 samples: two samples targeting the archael 16S gene and two samples targeting the bacterial 16S gene, each for one of the anaerobic cultures. Completing this large PCR task felt quite accomplishing. The gel taken after the PCR revealed that the archaea DNA degraded or the archaea did not grow much at all because very little 16S DNA appeared. For bacteria however, the 16S genes did appear, showing that some bacteria did grow in the anaerobic cultures.

            This week I also learned a new procedure that allows for the detection of the archael DNA in E.coli clone colonies. This method involves the use of DNA probes and development of film. A DNA probe is a sequence of DNA that is made to bind to target DNA. In our case, our probe was sequenced to bind to the archeal DNA. To apply the probes, we used a method of blotting where we used a circular piece of paper to pick up colonies from a selected agar plate. After applying the probes, we used a reagent that caused the target DNA and probes to produce light. We exposed film to the blots for a half an hour and over night in a dark room. The spots on the film that were dark show a positive hit. This tells us that there is archeal DNA in that colony of E.coli clones.

            Running a large PCR and learning to probe for DNA were two large tasks that consumed most of my week. After running the PCR, I feel a lot more confident about more PCR and about the goal of my project. The same goes for the blot making, a procedure I will be conducting throughout the next few weeks.