Taylor Hutcheson

Date:
April 28, 2017

Abstract
In our research, we gathered soil samples, picked out ciliates from these samples, and then extracted the DNA from the ciliates. Our objective was to extract DNA and use gel electrophoresis to determine the species that the DNA belongs to. Our gel electrophoresis results yielded no DNA, so our attempts to extract DNA were unsuccessful, therefore we were not able to identify a species based on DNA.

Introduction
Ciliates are microscopic single cellular eukaryotic organisms belonging to the phylum ciliophoran. They are characterized by their hair-like projections called cilia, which is where they get their name. Ciliates have two nuclei (one macro and one micro) and feed on small organisms such as bacteria through their oral grooves. They can reproduce sexually and asexually and play an important role in agriculture.

Materials/Methods

1. Non-flooded Plates
   1. Add 10 g of soil and add water, place under microscope and pick out ciliates.
2. Culturing
   1. Add picked ciliates to a medium of ceraphyll to encourage reproduction.
3. DNA extraction
   1. Transfer 300 uL of ciliate culture to centrifuge.
   2. Incubate for 30 minutes in a 56 C water bath to denature proteins.
   3. Boil for 8 minutes and vortex for 1 minute.
   4. Centrifuge to pellet cellular debris
   5. Transfer supernatant, that contains DNA, to microcentrifuge tube.
4. PCR and Gel Electrophoresis
   1. Denature proteins in a water bath of 94 C.
   2. 35 Cycles
   3. Denaturation: 94 C for 30 s
   4. Primer annealing 56 C for 20 s
   5. Primer elongation 72 C for 2.5 min
   6. Extension: 72 C for 5 Min
5. Gel Electrophoresis =
   1. Prepare agarose gel with wells for DNA
   2. Run electrical to separate DNA fragments.
   3. Used as a tool to show genetic diversity (Jousset 2010)

Conclusion/Discussion
Because our gel electrophoresis results showed no bands or fragments of DNA, we came to the conclusion that we were not able to successfully extract the DNA from our ciliate cultures. This is also shown by the low amount of DNA in the results from the Nano Spectrophotometer. This could be be a result of not following procedures, technical difficulties, human error, or contamination. This research is significant because it proves “most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments” (Kim 2009).

Literature
Jousset, A., Lara, E., Nikolausz, M., Harms, H., & Chatzinotas, A. (2010). Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil. Science of the Total Environment, 408(5), 1221-1225.

Kim, S., & Min, G. (2009). Optimization of DNA extraction from a single living ciliate for stable and repetitive PCR amplification. Animal Cells and Systems, 13(3), 351-356.

Shimano, S., Sambe, M., & Kasahara, Y. (2012). Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils. Microbes and Environments, 27(2), 136-141.

Taylor Hutcheson

Date:
April 20, 2017

Disclaimer:
As does happen sometimes in scientific experiments, our PCR procedure did not go as planned. Our class learned that the machine that was supposed to hold our DNA samples at 4 C overnight malfunctioned, holding our samples at 94 C – a much higher temperature, resulting in the evaporation of all of our DNA. As a result, we (or rather, the TA’s) had to recreate the tubes used in last week’s experiment.

Goals:

• Our objective was originally oriented on using our DNA after the PCR in a gel electrophoresis experiment so as to separate the DNA for identification, however, because this was no longer plausible, we focused on practicing gel electrophoresis until it was time to head to the computer lab and learn about how to create our posters.

Background:

Gel electrophorsesis separated fragments of DNA by size, with larger particles being closer to where they are placed and smaller particles traveling farther. The gel is placed in a salt-water solution so as to conduct electricity. The gel is stained by a DNA-binding dye and placed under a UV light, allowing the DNA to glow and become visible.

Procedure:

Gel Electrophoresis

1. The TA’s recreated our treatment groups for us, this time not taking a control group into account due to limitations on time.
2. After the TA’s recreated the mixture of Taq. Mix and EUK primers as according to last week’s procedure, we added the 5 uL of DNA solution, which we had preserved from our previous lab.
3. We then vortexed the tubes for about 30 seconds, so as to ensure the reagents had mixed and settled to the bottom of the tube.
4. The tubes were then sent again to another machine so as to run it through the process of heating and cooling (explained in last week’s lab).
5. Finally, we practiced loading the gel electrophoresis trays with faculty DNA.

Computer Lab

1. We reviewed how to create a proper scientific poster, learning that it is very important not to have too much text and to have many pictures.
2. We then began brainstorming with our lab partners regarding how to go about the creation of our posters and would information we would like to include.

Data:

This is the photo of the results of my group’s gel electrophoresis after the TA’s ran our DNA through gel electrophoresis.

Future Experiments:
Because we our short on time, our next and final lab will simply include our poster presentations. If given more time, we would most likely try to redo the PCR and gel electrophoresis experiments with more pure concentrations of cultured DNA.

Taylor Hutcheson

Date:
April 13, 2017

Goals:

• Prepare a PCR reaction using SSU ribosomal/universal primers (EUK). This will help to separate the cellular material from the nuclear material (though ribosomes may be an issue, as they have genetic material) in preparation for the gel electrophoresis.

Procedure:

1. The control group will have an addition of 5 uL of water, while the treatment group will have the addition of 5 uL of DNA. Other than this, the process is the same for the two tubes. The control group is there to ensure that there were no outside factors or extra DNA found in the nuclear material.
2. In the EUK tube, (mine labeled with a star), add 5.0 uL of DNA.
3. In the control tube, add 5.0 uL of water.
4. In both tubes, add 12.5 uL of Taq. Mix, 1 uL of EUK primers, and an additional 6.5 uL of water. After all this, the total volume of both tubes should be 25 uL.
5. Vortex both tubes for a short amount of time to ensure that all of the reagents mix and settle to the bottom of the tubes.
6. The tubes will now enter a thermal cycling profile.
   2. Initial denaturation: Heat the tubes at 94 C for 2.5 minutes.
   3. Now, the tubes will endure 35 cycles of:
      1. Denaturation: Heat the tubes at 94 C for 30 seconds.
      2. Primer annealing: Cool the tubes at 56 C for 20 seconds.

• Primer elongation: Heat the tubes at 72 C for 2.5 minutes.

1. Extension: Keep the tubes at 72 C for 5 minutes.

Future Experiments:
The next step will be to use the extracted DNA to perform gel electrophoresis, which uses an electrical current to distribute the nuclear material. From here, we will, in theory, be able to identify what type of ciliate the DNA belongs to by comparing it to the research of others.

Taylor Hutcheson

Date:
April 7, 2017

Goals:

• Extract the DNA of dense ciliate culture so as to prepare for the process of gel electrophoresis.

Procedure:

1. Transfer 300 uL – 500 uL of ciliate culture to microcentrifuge.
2. Centrifuge for 5 minutes at 6000 g, discarding supernatant.
3. Incubate for 30 minutes in a 56 C water bath, as this will break open the cells and denature some proteins.
4. Boil for 8 minutes in 100 C water bath.
5. Vortex for 1 minute.
6. Centrifuge for 3 minutes at 16000 g to pellet cellular debris.
7. Transfer supernatant with DNA in solution to microcentrifuge tube, without collecting the pellet.
8. Label this tube with the soil identified (mine was TDH06S17) and place it in an ice box.

Observations:
After checking to see if my ciliates had cultured, I was pleasantly surprised to find that, under 40x magnification with a compound microscope, there were too many ciliates to count, all moving around quite rapidly.

Future Experiments:
After obtaining the DNA solution, the next step will be to prepare PCR reactions and gel electrophoresis so that we can identify the type if ciliate in my soil sample.

Taylor Hutcheson

Date:
March 30, 2017

Goals:

• Continue searching for ciliates within the non-flooded well plates with the intention of extracting ciliates from the solution and placing them in Cerophyll media so that they may culture.
• The goal after obtaining cultured ciliates would be to extract their DNA so as to determine which type of ciliate the organism belongs to.

Procedure:
This lab is almost identical to the previous lab.

1. Observe the non-flooded plate under both a dissecting and compound microscope, searching for ciliates to cultivate.
2. When able to extract ciliates, use a pipette to extract about 2 uL of the ciliate and its surround solution, and place in a 24-well plate with 1000 uL of Cerophyll.

Observations:
After having no luck retrieving ciliates from the plate under a dissecting microscope due simply to how fast they swim, I decided to use a compound microscope. I took a small sample of water from the plate and placed it on a concavity slide, observing it at 4x, 10x, and 40x magnification. It was not until 40x that I found multiple small ciliates. I realized, after backing up back to 10x magnification, that I could observe the ciliates causing small solid particles such as dirt and shrubbery to move with their motion through the solution. I then followed the procedure and placed multiple of these ciliates in well A1 of my 24-well plate with the Cerophyll media.

Future experiments:
If, by next week my ciliates have cultured, I will be able to move on to the DNA extraction of the dense ciliate culture.

Taylor Hutcheson

Date:
March 23, 2017

Goals:

• Complete testing and analysis for the metadata of the soil sample.
• Continue the process of extracting ciliates from the non-flooded plate.

Procedure:

1. Use drops of water from the non-flooded plates to determine the pH of the soil.
   1. Using a plastic pipette, drop some of the water from the plate onto a piece of pH paper.
   2. Compare the color on the paper to that of a key.
2. Weigh the dry soil sample and compare it to its weight last week.
3. Continue searching soil samples for ciliates.

Observations:
After allowing my soil to sit in the non-flooded well for a week, I had high hopes that the ciliates would come out. Towards the beginning of the class period, I did not find a single organism. About an hour in, I finally found a very small, circular ciliate moving very quickly through the water-soil solution. I would then find one about every 30 minutes after that, but always moing too quikly to pluck from the solution using the pipette. I found these ciliates in the center of the plate, always near a body of soil rather then in an ocean of water (or what would be an ocean to a ciliate). Once, on the outer edge of the plate, I found a relatively larger and longer ciliate. I still good not harvest a single ciliate. I even tried taking small droplets of about 400 uL of the non-flooded plate’s water, and analyzing just those droplets.

Data:

1. pH: 6.0 – slightly acidic
2. Soil type: Silt loam
   58. Silt 58.8%
   59. Sand 29.4%
   60. Clay 11.8%
3. Dry weight of soil: 4.2 grams
   1. Water made up 0.8 grams of the soil’s weight

Future Experiments:
From here, it will be beneficial to continue the search for ciliates. I think a strategy I will pick up next lab will be checking a sample of soil under a compound microscope.