Week 8: New projects and end of ESP deployment

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.  Even one of the known standards we were testing came back with the same message, so overall, it was not a good run.  I also ran a gel of two of the samples to check for any damage to the DNA; upon looking at the gels under UV light, we determined the samples were indeed viable.  This week we had a lab meeting in which we talked about collaborating with the adjacent lab on a project that would attempt to classify microbes in various locations in the estuary with a new sequencing technique; in this way, we could provide key samples for this project.  In preparation for this new project, Lydie and I outlined a plan of action, and brainstormed what samples we would use, making sure to cover a wide range of conditions (different locations, low/high dissolved oxygen, ETM/non-ETM, sediment/water, etc.).  We are going to have to run these samples through PCR in order to amplify the DNA, so to prep for this I did a practice run with four of the sediment samples; after PCR, I ran the samples through gel to make sure there was enough DNA present for sequencing.  However, the first gel showed up very faint under the UV light, so I re-ran the samples the next day in order to produce a better image.  The second run looked much better although two of the samples seem to need to be adjusted because their bands did not show up as strongly.  I also ran two of the sediment samples through a thicker gel, whereby I extracted the DNA from the gel and purified it, which involved cutting out part of the gel saturated with DNA, incubating the gel segment with a buffer to dissolve it, and then a series of adding more buffer and TE in order to purify the DNA and several rounds of centrifugation to obtain the final product.  I then ran the purified product through gel, and it showed up well under UV light, so it seems the purification was a success.  Later in the week, we received a new PicoGreen dsDNA Assay Kit, so I prepared the same samples as I had earlier in the week for quantification.  The first run already appeared to go much better than the previous run; all of the samples except for one provided concentrations upon nanodropping.  However, many of the values seemed to be close to the limit of the standard curve, so we decided to dilute some of those samples down and run them again in order to get more accurate readings.  So I prepared new standards and sample solutions and ran it again; a few of the samples had better readings, but a few still seemed to be reading at the limit of the standards, so next week we may have to dilute these samples further and run them again.  Also, all through the week I had been inputting the data from the ESP deployment; Lydie received an email every time a sample was collected, whereby she forwarded it to me so I could record the various data (date, times, turbidity, dissolved oxygen, salinity, temperature, etc.) into the lab notebook and sample list.  This week marked the end of the ESP deployment, and Friday we were excited to receive the pucks from the ESP, so next week we can start extracting those samples.