Week 5: Finally Getting Results

This week I once again tried to get some decent qPCR results.  My first run did not go well at all, and I ended up with my worst efficiency to date.  When analyzing my data, it seemed that my pipetting was fairly accurate, but the cycle threshold values were consistently off, indicating a problem with the preparation of the standard solutions rather than error in pipetting technique.  However, I made new standards on Wednesday, and after great anticipation I was finally rewarded with an efficiency value of 99%.  This was exciting because it meant that I could finally take another stab at running some of our water samples in order to detect archael amoA genes, which are then used as biomarkers for determining AOA abundance.  We are hoping to track AOA abundance throughout the estuary and examine how differenct conditions (such as low dissolved oxygen, tidal influences, ETM, etc.) affect AOA abundance as well as discover how AOA get into the estuary (i.e. are they free-living or are they particle-attached).  I ran a full plate of samples, and I was very excited to get good efficiency again (97%), but even more excited about the fact that I finally had real data!  We are still in the process of putting this data together into graphs to have a better feel of any conclusions that can be made, but already it seems that there are definitely some patterns arising that will be interesting to investigate further.  While finally getting results was by far the highlight of the week, I also made stock solutions of all the reagents needed for RNA extractions that we will perform sometime in the near future and tried again to get decent readings from the fluorospectrometer.  This time we used a linearlized plasmid that Lydie prepared in April to prepare dilutions of known concentration to test on the fluorospectrometer to see if the readings would match the values we had already calculated and knew to be correct.  However, the values did not match in the end, and we are still trying to figure out whether it's a problem with the standards, the reagent kit, the preparation of the samples, or something else entirely.