Week Six: The qPCR finally worked

After one final blank test with the 353-487 primers in which I finally saw no contamination, I was able to use the primers on our archaeal sediment samples and the results were great (Figure below). All the samples showed good amplification and the data we were able to get showed very interesting graph of the abundance of the amoA gene for the sites view graph

I also preformed a regular PCR amplification on the archaeal sediment samples using the Arch-amoA primers in order to see if the late peaks we had seen were producing a separate gene fragment then those who showed peaks at the correct temperature. The gel showed that all the samples were amplifying the correct size fragment despite were the peak fell on the qPCR graph.

I also preformed a qPCR to check if the RNA samples still had significant amounts of DNA and since there was a lack of amplification I will proceed to preforming reverse transcription on them next week in order to create a cDNA library.

Since the touchdown qPCR had failed to start I preformed a normal touchdown PCR on the sediment bacteria samples to see if the protocol would actually decrease the non specific binding seen in previous amplifications while providing better amplifying the 500bp amoA fragment. The PCR showed good amplification for all tested samples for the 20x dilutions so I am in the process of figuring out the correct way to program a touchdown qPCR.

This week I also was able to present all of my data during a lab meeting and receive feedback on plan how best to proceed as well as on how to improve my presenting skills. I helped back for the sampling trip we will be embarking on next week.