Kiley Seitz's blog

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.

I started off this week by running a touchdown qPCR with the archaeal primers to see how similar the numbers were when compared to the qPCR, that way we would know if the we numbers we get with the touchdown qPCR for bacteria are an accurate representation of what we would see with a normal qPCR. Although the numbers for the touchdown qPCR were higher and statically different it was not by a great amount.

This week we went back to Astoria to collect more samples. This time we got them from ten of our sampling sights and for two of the sites we took a sample from the top layer and the bottom layer from two sites.  We also prepared the ESP for deployment on Wednesday and took it out to Sat 3 to set it up.

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

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

This week I had some problems with the qPCR I was preforming.

On Monday Lydie and I went to Astoria in order to run maintenance on the ESP.  The amount of water being flushed through the syringe during the last deployment of the ESP did not match the volume that Lydie had expected to be flushed. This suggested that there could be a problem with the syringe but upon examining the ESP we found that the real problem was that the bag filled the flush solution had a kink in the line that prevent the liquid from flowing to the syringe. By simply moving the bag to a higher point on the ESP we were able to rectify the problem. 

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.

After getting a tour of the building and meeting all the new interns, I went for coffee with Lydie Herfort to talk about what had been done with the ESP column samples and the soil samples since my visit this spring. We went over some slides and graphs that she had recently made of the sampling times of the ESP and we decided that I should first start on extracting DNA from the recently collected soil samples. Using the FastDNA Spin kit, which I had not used before, Lydie and I extracted the first two samples together to make sure I didn't have any problems using this particular kit .