Week Two: qPCR attempts

This week I tackled qPCR, starting with the DNA that I extracted from the soil samples last week, as well as some that Lydie had already extracted, in order to get an idea of the amount of Ammonium Oxidizing Archaea. My first attempt at qPCR showed half of the samples with skewed results and, as it turned out, all of the samples that gave pour results were the ones I had extracted. Between that and messing up to the standards the run had to be completely disregarded.

Lydie had me analyze the data anyway, just so I could practice, and in doing so I was able to determine the problem with my samples. Instead of diluting the samples 50x, like Lydie had done with hers, I had made 50X solutions with all of mine that allowed for inhibition in the samples.

Overall that was great news and after preforming correct dilutions and getting new water to dilute the samples in, the qPCR worked beautifully.  This means, among other things, that I did not mess up the extractions, which is very good to news for me.

Only two samples still had bad results and both were from the same sampling site. Since past results has shown that this site has a large amount of Ammonium Oxidizing Archaea, we are going to create a dilution series in order to see if this site is still experiencing inhibition.  Overall I am very please with the data I got this week.

I also stumbled across something else this week. In doing a RNA extraction I spilt the second extraction for one sample and was forced to take a third. I expected the nanodrop results for this sample to be considerably worse then the other samples since, in theory, by the second extraction there should be a large decrease in the amount of RNA collected. Instead the results for this sample was purer then those of the other two samples were, leading me to wonder if we shouldn’t be taking a third extraction from all of the samples. I plan to test this theory but until then we will continue extracting RNA as planned.