Capture, Culture, and DNA Extraction of a Novel Soil CiliateJay Winn Ashley Washington

Baylor University

Abstract

Introduction

Methods (Continued)

Discussion

Conclusion

References

Results

The purpose of this experiment was to learn various laboratory processes such as DNA extraction, polymerase chain reaction, and gel electrophoresis and to possibly identify a novel soil ciliate. To do this, we “adopted” a soil sample collected near the Founder’s Mall Flower Beds in Waco. We analyzed our soil in order to find, isolate, and culture ciliates. We then extracted the DNA from our ciliates, and used a Polymerase Chain Reaction to produce copies of the ciliate DNA. We then used gel electrophoresis to separate the DNA fragments. The experiment did not produce any positive results, most likely because of protein contamination.

2. CULTURE SOIL CILIATES• Weigh 5 grams of wet soil• Obtain the dry weight of the soil data and record• Use a pH litmus paper to measure the acidity of soil sample• Calculate the water content• Add water into the falcon tube to the 10 ml mark• Add a suspension drop into the falcon tube• Let the solution content settle to produce visual lines of

where the clay, silt, and sand levels are located

The goal of this experiment was to identify soil ciliates that were found by Founder’s Mall in Waco, Texas by extracting their DNA. We were unable to successfully extract and analyze the DNA. This was most likely due to high protein contamination which may have had an effect on downstream applications-like electrophoresis- that used the nucleic acid samples. Our data serves to help future scientists avoid our mistakes and thus, facilitate their research concerning DNA extraction ad soil ciliate identification.

Figure 1. Picture above shows the Location of Soil Sample

Figure 2. Name of Chart

1. Lynn D. (2009), Ciliates, Encyclopedia of Microbiology. 590p.

2. Foissner W. and Berger H. (1996), A User-Friendly Guide to the Ciliates (Protozoa, Ciliophora) Commonly Used by Hydrobiologistsas Bioindicators in Rivers, Lakes, and Waste Waters, with Notes on their Ecology. Freshwater Biology, 35: 375-482, 376 p.

3. Moore P. (2005), PCR: Replicating Success, Nature, 435: 235-238, 235 p.

Figure 12. The above table shows the Soil Metadata

Figure 13. The table above shows the Nanodrop Results

Figure 14. The table above shows the PCR Assay Table

Figure 15. The image above shows the results of gel electrophoresis. None of the wells (Control EUK or EUK) seem to contain any DNA, except for the ladders which can be seen at the far most left and thefar most right sides.

• The low nanodrop ratios stated in the nanodrop resultsindicate that the samples are protein contaminated.

• In the PCR, the treatment groups contained more DNA and less water. Ideally, there should have been a greater concentration of ciliate DNA in the Treatment Group after the PCR took place.

• The electrophoresis results produced by the samples were negative, so they cannot be compared to other known results for ciliate identification.

Figure 2. Picture above shows Soil Collection

Figure 3. Picture above shows a Dissecting Microscope

Figure 4. Picture above shows thenon-flooded plate

Figure 5. Picture to the right showspH litmus paper. The color shows that the pH of our soil was 6.0.

Figure 5. Picture above shows the examplesof soil samples in falcon tubes with varyinglevels of sand, silt, and clay

Figure 6. Picture above shows varying levels of Clay, Silt, and Sand of a Soil Sample. Our soil sample did not show this clear separation.

1. COLLECT SOIL AND OBSERVE CILIATES• Choose an area of your choice to find ciliates in the soil sample• Under a dissecting microscope, extract the ciliates from the non-flooded plate

3. DNA EXTRACTION• Isolate the ciliate in a solution of Cerophylland distilled water• Use the modified Chelex Protocol

• Transfer ciliate culture to centrifuge• centrifuge at 6000g for 5 minutes and dispose of the supernatant• Add 200 microliters of 5% Chelex solution• Vortex for 1 minute• Incubate for 30 minutes in a bath water and boil for 8 minutes in the 100

degrees Celsius• Vortex again but in 16000g for 3 minutes and extract the supernatant

Figure 8. Picture above shows the centrifuge in the DNA Extraction protocol

Figure 9. Picture above shows extraction of the supernatant

4. PCR METHOD

•Label 2 tubes: 1 control and 1 treatment (i.e. 6C and 6CC)

•Perform the PCR method at 25 microliters

• 5 microliters of ciliate DNA into a TAQ mix (12.5 microliters) for treatment

• Put 1 microliter of the 10 micromolars primer in bot the control and treatment

groups

• Add 6.5 microliters of water to treatment group and 11.5 microliters of water

into control group

• Vortex the solution

PCR METHOD (CONTINUED)

•Perform Thermal cycling profile

• Initial denaturation: 94 degrees Celsius

• Denaturation: 94 degrees Celsius (30 sec)

• Primer annealing: 56 degrees Celsius (20 sec)

• Primer elongation: 72 degrees Celcius (2.5 min)

• Extension: 72 degrees Celsius (5 mins)

• Hold at 4 degrees Celsius

•Gel Electrophoresis

• Repeat the PCR method due to thermocycler malfunction

• Using the DNA, begin to load the gel

• Observe the transfer of the DNA through the gel

Figure 7. Picture above shows our soil sample, which has no clear separation

Figure 10. Picture to the left shows a vortex

Figure 11. Electrophoresis

Methods

Capture, Culture, and DNA Extraction of a Novel Ciliate discusses DNA Extraction as a common routine for biological studies such as molecular identification. The ideal route is to optimize DNA yield and minimize DNA degradation. We want to experiment on a particular soil and use a biological method that can break down the soil into its DNA components and apply the Gel Electrophoresis method to illustrate the movement of DNA due to current produced by electron carrying. The importance of soil ciliate discovery is to grasp a better understanding of organisms that are free living and aquatic can effect biodiversity of an environment and if it has a positive, negative or no impact if the organisms sharing an environment with them. The data used for experimentation was from another student, Elizabeth McElroy because our soil samples did not contain any ciliates.