Week nine: Starting cloning

This week I started cloning. The first thing I did was preform a normal touchdown PCR of the sediment samples using the bacteria primers. Next I made LB agar plates for culturing later on in the process. I then preformed a purification step on the PCR product in order to get the extra protein and enzymes out before preforming the TOPO cloning process and freezing the product for this week when we will continue the process.

This week I also ran the cDNA that I created last week but the majority of the samples gave strange peaks that we believe is consistent with DNA contaminates and low levels of cDNA. In order to fix this I re-preformed the RT-PCR using a higher quantity of RNA in hope of achieving a better product. This week I will run the samples in order to see if this is true.

The water bacteria samples are still giving me trouble with a second band appearing whenever I try a protocol with multiple temperatures. I will attempt to preform a normal PCR with an annealing temperature of 59 and one with 60 degrees Celsius to make sure a simple amplification will only produce one band.

This week we also toured the OHSU main campus, which was a great experience, and the view was amazing. I was also able to give a practice talk for my presentation next week that was nice practice and helpful in letting me understand the larger implications of my project.