Madison Hibshman's blog

I started out the week running some sediment DNA samples through the fluorospectrometer to try to accurately quantify the DNA.  The fluorospectrometer has proved a lot easier to handle ever since we got new chemicals, and all of the samples but two showed readings in the range of the standards.  I also ran a PCR of some samples of different dilutions to see which dilutions we would use for sequencing; after PCR, I ran the samples in a gel, but we could not verify which bands were indicative of the correct base pair when using the 1 kb ladder as a reference.

On Monday, I attempted to use the fluorospectrometer to quantify DNA concentrations in various samples as well as standards of known concentrations so we could check their accuracy.  However, I ran into many difficulties when trying to take the nanodrop measurements; when it came time to run the actual samples, an error message popped up for several of them reading "the measurement concentration is outside of the range of reference or standards", and so the concentrations could not be determined for these samples.

This week I once again started out with a qPCR of the same plate I ran last week because the efficiency had been too low.  I made new standards and ran a full plate, yet unfortunately, when I analyzed the results the efficiency was still too low to my frustration.  The next day I used Kiley's standards for the same plate of qPCR because her standards had yielded good efficiency in her last run.  The data looked good upon analysis although some of the lower standards had to be thrown out.

This week was by far the busiest week of my internship thus far.  Monday and Tuesday I ran full plates of qPCR with variable results.  The first run displayed an efficiency of 107%, which was within the accepted range.  In effect, this data was viable, and displayed some interesting 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%.

When I analyzed last week's qPCR data, I found that the efficiency was only 85%, which was below the value we were hoping for. So this week I got to practice some more qPCR, and hopefully when I analyze the most recent data, my efficiency values fall in a more acceptable range.  We also started planning for RNA extractions next week, which involve a lot of solutions that need to be prepared, so I took the time to plan out all of the solutions and calculate all of the necessary volumes and such.

This week Lydie was absent, so I performed DNA extractions tediously on soil samples collected by Kiley Seitz for the majority of the time.  However, Wednesday our normal routine was upturned as we took a trip to the coast to various sites in order to collect new soil and water samples for analysis.  As much as I was excited to get out of the lab for a bit and get some field experience, waking up at 2:45 in the morning was not ideal.  However, at 3:30 Wednesday morning, Lydie, Kylie, Kylie's intern Isla, and I were off to the coast.

After copious amounts of DNA extractions the first week of work I was ready to start some new projects.  This week I spent a few days running gels, which ended up not yielding the best images; the resulting bands were extremely faint if apparent at all in many of the runs due to the extremely low concentrations of DNA in the collected water samples.  However, we were able to identify a correlation between the quality of the images (whether there were bands present) and the quality of the filter from which the sample was collected.

My name is Maddie Hibshman, and I am working in the Simon lab under the mentorship of Lydie Herfort.  This summer I will be examining the microbial dynamics in the Columbia River Estuary by an autonomous adaptive sampling approach.  Lydie quickly got me acclimated to the work place and gave me an abundance of literature to read up on to obtain a more detailed understanding of my project for the summer.