Week Five: Problem solving

This week we attempted so solve all the problems we had been having with the qPCR for our different samples:

For the water column Bacteria samples we preformed a gradient qPCR in order to find the optimal temperature for the reaction. When none of the temperatures produced the peaks we were looking for we ran the PCR results out on an agarose gel to see if they had the correct size band. The Gel showed that for the higher temperatures the correct gene was being amplified so we have decided that we would clone the gene in order to create primers that are more suited for our samples. 

We then attempted to do the same process for the sediment Bacteria samples. Once again we preformed a gradient qPCR and then ran the results out on the gel. This gel however, had no temperature that could give us a single band at the correct size so we choose the temperature that had what appeared to be the strongest results and decided to preform a touchdown qPCR in order to decrease the amount of nonspecific binding that was occurring. The problem with this seems to be an inability of the qPCR machine to do a touchdown qPCR so a different solution will need to be tried.

For the Archaea sediment the new primers we ordered came in but when we preformed the qPCR with them we still saw contamination in the blacks. In order to rectify this all equipment was thoroughly cleaned and new supplies of everything were attained in order to eliminate the contaminate but when a blank test was run their was still contamination. We then ran out the blanks to see if the contamination was actually the gene we were looking for or if it was nonspecific and saw that the contamination was indeed the amoA gene. We then had Mouzhong attempt the blank test to compare her supplies to mine in order too see if the contamination was in the material or in the technique. This qPCR reveled that there is indeed something in my supplies that is causing the false positive so we will be performing more test qPCRs in order to isolate it. 

I also extracted the last of the RNA from the filter samples and preformed a purification step on all of the RNA samples in order to remove all traces of DNA form them.