Problem solving with contamination and data entry

Since my last post, I have completed more PCR and gel electrophoresis with the adult ticks. Everything was going very smoothly until a couple of days ago when I ran a gel and found every single tick came out as positive for the pathogen Ehrlichia chaffeensis. This seemed abnormal and quite frankly, alarming. If all of the tick extracts that were used in that agarose gel were in fact positive, that would have put the prevalence for 2016 above 20% which is much higher than 2017 and 2018. A difference that high did not seem right to me so I knew the odd results must have been due to contamination that occurred along the way. So, I began trying to figure out what to do and after working with my professor we came up with the solution to use new primers. These new primers that I have begun using are longer in base pairs. Therefor, any contamination that could have occurred with the old primers gets discounted as the new primers attach to a longer section of the DNA. I re-ran the contaminated agarose gels and they came out normal again, giving me only 2 positives instead of 32.

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Vibrio Fischeri Wrinkly Spreader vs. Smooth Morph Growth So Far

So far this summer, I’ve been able to collect data on V. fischeri growth over a salinity gradient spanning most of the range between zero percent sodium chloride and nine percent sodium chloride with intervals of 0.2 percent sodium chloride. I currently have data on zero, one, two, three, all the way through nine percent and I’m working on finishing the intervals within that range.

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Seeding and Transfection for 2nd and 3rd Trials

Yesterday, I successfully seeded HeLa cells into a six-well plates for my 2nd trial. Before seeding, I ran into a small issue. Over the weekend, my HeLa cells have multiplied so much that they are now growing on top of each other. With my PI’s help, I proceeded with trypsinizing the overgrown flask, but this time I diluted the cells with double the amount of medium I was supposed to use. This technique has yielded satisfactory results, as I was able to see a nice population of cells in each of my wells under the microscope this morning. After viewing my six-well plates, I continued on with transfection. Similar to my 1st trial, I am now transfecting my cells with PMT2, MK-STYX, and F1 (active mutant of MK-STYX that has dephosphorylation capability). Although the 1st trial from last week was successful, I did notice a decrease in transfection efficiency. This might have resulted from the fact that I transfected the cells 22 hours after seeding. To increase my transfection efficiency, I decided to transfect the cells ~18 hours after seeding this time. I’m hoping that I would be able to see more cells transfected than last time so that I would be able to see more of the effects that MK-STYX exerts on the localization of the autophagy regulator TFEB (transcription factor EB). Today, I will also be seeding another six-well plates from a different flask. I have already viewed the flask this morning and there is a nice population inside the flask, so I will only need to trypsinize the cells and will not need to dilute them. At this point, I am paying much more attention to every step of my procedure, making sure to reduce any errors that might possibly contribute to not providing the maximal data.

Combo: the mediun that keeps zooplankton happy

The preparation for my common garden experiment for zooplankton Daphnia pulex and Simocephalus vetulus is going well. To make sure my beloved cute zooplankton can stay very happy during my experiment and ever after, my professor and I decided to make a favorable and standardized medium for them. Combo medium is our choice. It is composed of 25 chemicals and is proved to support both algae and zooplankton culture well. As my professor said, “Combo lab is fancy lab”. This medium will make our lab a cool lab while my pulex babies thrive in it.  I’m very glad to report that our first batch of Combo made my pulex very happy.

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More on Troubleshooting and 1st Successful Trial

With all my cells dying lately, I have began to feel that I might not be able to see any successful experiment during this summer. However, a last minute consultation with my PI has allowed me to alter a small portion of my protocol to yield a successful experiment. With my past experiments, I have been using DMEM with 0% FBS as the medium for my HeLa cells when I serum-starve them. After seeing that my cells keep dying after less than 18 hours of serum-starving, my PI has advised me to switch my choice of medium. So instead of using DMEM with 0% FBS, I am now using DMEM with 0.1% FBS. This gives my HeLa cells just a bit more nutrient and allows them to survive during the overnight starvation period, as well as go into autophagy mode. This worked out perfectly, as my cells survived after an overnight period of starvation and I was able to continue on with fixation. During fixation, I also made sure that my cells remained hydrated. I have been doing this by not completely aspirating away all the D-PBS during my wash steps. In the past, I have noticed that not all my cover slips were completely submerged in D-PBS when I am adding in more D-PBS after aspirating them out. This time, I also made sure that each coverslip are entirely submerged in the D-PBS at all times because my cells are facing up, so it is crucial that the coverslips are submerged. So far, these small adjustments have allowed my cells to survive and give rise to my 1st successful experiment of the summer. I was able to see the results of TFEB localization in HeLa cells upon serum-starvation and in the presence of MK-STYX as of Friday last week. I am extremely happy with the data that I’ve obtained and am looking forward to having more successful experiments this week.