Investigation and DNA Characterization of Soil Ciliate Diversity Angelica Anzures and Kasia Cross Abstract Methods Methods Results Introduction: The soil studied in this lab was taken to investigate and classify the ciliates found. Methods: Methods employed in this experiment included the making of non-flooded plates, the capture and culturing of ciliates, PCR, Gel Electrophoresis. Results: Experimental results revealed that soil sample A contained more ciliates than soil sample B. NanoDrop outcomes for ciliate DNA from both soil A and B indicated protein contamination. DNA characterization through PCR and gel electrophoresis methods yielded inconclusive results. Discussion: The possible culturing and DNA amplification of a nematode in both soil A and B samples, along with several instances of equipment failure, likely contributed to inconclusive DNA characterization of soil ciliates.   Non-flooded plates: A non- flooded plate was made of each sample. Sample A lacked ciliates and was replaced. Both of the samples were stored away in Isotemp Incubator. Soil information was recorded (table 1).​             Preparation for PCR: A modified chelex ​extraction protocol was followed. Universal PCR assay was preformed by placing 2xMaster mix, ciliate DNA, and the primers into PCR tubes (Table 1). The control group did not contain DNA. The tubes were then stored and Baylor faculty carried out PCR. It was discovered that the thermocycler had malfunctioned and was operating at 94 degrees Celsius, causing the reaction mixtures within the centrifuge tubes to evaporate. Faculty of the Biology Department added water to the evaporated mixtures and ran one positive control on a gel. The PCR preparation process was repeated.​ PCR & Gel Electrophoresis​: The process of PCR was carried out by faculty and involved the programming of a thermocycler. To run Gel Electrophoresis, 5 uL of the Quick-load 1kb DNA ladder was added along with 1o uL of each PCR product. The gel was run on 90 t0 100 volts. Another mishap occurred with the gels and the Gel Electrophoresis process was repeated by department faculty, with two combs per gel, and imaged using UV light. Culturing of ciliates​: After a week’s time numerous ciliates were found from sample A and ~8 ciliates in sample B. Both samples contained a nematode.​ Preparation for PCR: It was discovered that the thermocycler had malfunctioned and was operating at 94 ∘C, causing the reaction mixtures within the centrifuge tubes to evaporate. The final concentration of each primer in the reaction mixture was 0.4uM. PCR​: Another mishap occurred with the gels​ and the bands of DNA did not show 1800-1900 base pairs (fig.6).​  Figure 2: Location where samples were collected. Sample A on the right and sample B on the left. Introduction Ciliates also play an important role in nutrient cycling by feeding on bacteria and releasing nutrients to primary producers [1]. Even slight changes in the environment can impact the growth of ciliates because they are crucial to many soil and aquatic ecosystems and have been suggested to act as bio-indicators of environmental stress [2].  The soil studied in this lab was taken to investigate just a few types of ciliates within the United States. Genetic components of the ciliates investigated are the main criteria for classification of the unknown ciliates. The primers used during the experiment indicate approximately 1800-1900 base pairs should appear on the Gel electrophoresis.​ Figure 6: Gel Results Results Discussion The inconclusive results of PCR and gel electrophoresis may be attributed to several sources of potential error in this experiment. Nematodes present in both samples may have contributed to the low 260/280 ratio, indicating protein contamination. Human skin cells contaminated the DNA. Table 1: Soil metadata plate that contained 900uL Cerophyll and 100uL water and cultured for a week. Culturing of ciliates : If ciliates were found in the non-flooded plates after a week’s time they  were captured and cultured. Ciliates were extracted using a micropipette and placed into designated wells in a 24-well. Acknowledgments A special thank you to Dr. Tamarah Adair, Michael Davis, and Sami Marley, the instructors that helped guide and execute the project and to Josie Minick for providing soil sample A. Figure 4: The estimated location of sample A and calculated location of sample B. Soil collection​: The water content of sample A(29%) was similar to the percent water in the original sample (20%) which was classified as a clay (fig 3).  Non-flooded plates​: Initial observations of the sample non-flooded plates revealed a high ciliate density in soil sample A and a low ciliate density-around 2 cells-in soil sample B. After 24 hours soil sample B had a notably higher density of cells were observed (~15). Methods Soil collection: Soil samples were​ collected from Waco wetlands and the Mayborn​ Museum. Water content, soil composition, and Ph were determined. Once percentages were calculated, a Soil Texture Triangle was used to determine the type of soil.   EUK Control EUK Tube # 4A 4AC 2xTaq mix (ul) 12.5 DNA (ul) 5 10uM EUK Primers A & B (ul) 1 Water (ul) 6.5 11.5 Total Volume (ul) 25 References [1]  B.S. Griffiths, Mineralization of nitrogen and phosphorus by mixed cultures of the ciliate protozoan Colpoda steinii, the nematode Rhabditis sp., the bacterium Pseudomonas fluorescens, Soil Biology and Biochemistry 18 (1986) 637-641.​ [2] Lara, Enrique, and Dimaris Acosta-Mercado. "A Molecular Perspective on Ciliates as Soil Bioindicators." European Journal of Soil Biology 49 (2012): 107-11. Web.​ [3] Raza, Azra, and Naomi Galili. "The Genetic Basis of Phenotypic Heterogeneity in Myelodysplastic Syndromes." Nature Reviews Cancer 12.12 (2012): 849-59. Web.​ Figure 1: Sample B tube. Table 1: The components of the mixture inside the PCR tubes.