April 27, 2017
Lab 15: Final Notebook Post
Goals: The process of ciliate discovery is essential in learning about the diversity of soil in our world. The present experiment aims to teach students the scientific process of ciliate discovery and classification, through the soil collection, DNA extraction, Polymerase Chain Reactions, and Gel Electrophoresis. Because of malfunctions with the procedure machinery, the results were inconclusive. It is important to educate students about this process, so that they can continue discovering new ciliates to contribute to the current knowledge of biodiversity.
Background: Over the course of the semester, students in the CILI-CURE laboratory have learned important techniques in biology research, acquiring skills in micro-pipetting and culturing. Students practiced researching and reviewing scientific literature to apply to the growth mindset standard in the classroom. In the laboratory, students focused on the diversity of soil ciliates, which lead the path to collecting soil samples from different areas in Waco to identify different ciliate species.
Methods:
Weeks 1-2
In the first two weeks, soil samples were collected from locations in Waco. 10-15g were observed in a non-flooded plate using a compound scope. Observations were recorded. 5.0 grams of the wet soil were stored and in the second week, the soil was further investigated. Additionally, 5 mL of the soil samples were added to falcon tubes and vortexed. The following week, the water content, percentages of sand, silt and clay, pH, latitude/longitude, and temperature of sample site were recorded.
Weeks 3-4
Using the non-flooded plates from week one, ciliates were extracted and cultured in 24 well plates with 900 microliters of Cerophyll media. If ciliates were not found, students used other’s soil samples. In the fourth week, a modified Chelex extraction protocol was used to extract ciliate DNA from the 24-well plates. Students added 5% Chelex to 500 uL of centrifuged ciliate culture, therefore protecting the DNA against enzymatic activity. The microtubes were then incubated at 56℃, boiled at 100℃, vortexed, and centrifuged. Remaining supernatant with DNA was transferred to new microtube.
Weeks 5-6
In week five, Polymerase Chain Reactions (PCR) were performed on previously collected DNA samples using SSU ribosomal and universal primers. A reaction mixture was created using 5 uL of master mix, 1 uL of primer, and varying amounts of water. Unfortunately in week 6, the thermocycler broke and overheated the DNA samples, causing them to evaporate. Process was repeated to test new DNA samples. Agarose Gels were made by mixing 40 mL of Tris acetate EDTA with agarose gel, and mixing them with 2 uL of ethidium bromide. Gel electrophoresis box was filled with agarose gel and allowed to solidify. Gel Electrophoresis was performed by adding 10 uL of each PCR product to the wells and turning the power supply to 100 volts. DNA was viewed with UV light.
Results:
| Soil Metadata |
AMJS1704 |
KACS1704 |
WBKS1704 |
| pH |
8 |
6.5 |
7.5 |
| Soil Classification |
Silt |
Silt Loam |
Sandy Clay Loam |
| Percent Water Composition |
29.4 % |
8 % |
18 % |
| Description of Location of Soil Collection |
Collected outside of the Baylor Science Building on Baylor Campus, in grass with no trees or plants near the site (Waco, TX) |
Collected outside of “Bear Park” on Baylor Campus, an area where grass was not growing (Waco, TX) |
Collected at Cameron Park, near a large tree and the Brazos River (Waco, TX) |
| Ciliates Found? |
Yes |
Yes |
No – used AMJS1704 sample for the rest of experiment |
Conclusion:
Throughout the semester, students in the CILI-CURE laboratory have learned to pick and culture cells to begin the process of ciliate discovery. Over the course of several weeks, soil collections were performed and DNA was extracted from the ciliates found in the soil. Polymerase Chain Reactions were performed, with the intention of Gel Electrophoresis. Due to malfunctions with the thermocycler in the PCR process, the results were inconclusive. The thermocycler froze, thawed, and heated the DNA too many times, causing damage and evaporation to the DNA’s composition. Additional sources of human error would include miscalculations when creating the PCR master mix. The pre-mixed primer could have been incorrectly mixed or the DNA could have degraded prior to the PCR protocol.
Ideas for Future Experiment:
I think it would be a really good idea to start this whole section of the lab sooner so that there is more time to collect an adequate amount of samples and have enough time to pick and culture.
I also think it would be beneficial to create a ciliate map for the city of Waco before we travel all over the world! I not only believe this will yield better results but also increase the level of responsibility and level of pride, blossoming young scientists have! What better way to make a difference in the world than by starting in your own backyard.
Lab 14: Real Life Science
April 20, 2017
Lab 14: Real Life Science
Goals: The purpose of this lab was to practice running DNA Electrophoresis on our prepared PCR samples. We also have to remake PCR samples.
Background: Unfortunately, the thermocycler that held our samples malfunctioned and evaporated our DNA! However, our instructors are the best and prepared the experimental PCR tubes, so that we only had to add our DNA.
Procedures-1: Redo PCR
- Add 5 microliters of DNA to prepared PCR tube.
- Label tube with your identifier.
Procedures-2: Gel Electrophoresis
- Load 10 microliters of failed DNA sample into well of 1.5% Tris-acetate-EDTA gel. *REMEMBER* Wells in gel should be closest to the negative cathode!
- Place the lid on the box, turn on power, and run supply at 90-100 volts
- Allow loading dye to run approximately halfway across gel; turn off power
- Image gels with UV light
Observations:
After what happened with the thermocycler, we will not know what ciliates we have by the time our next lab meets. Fail to succeed none-the-less!!
Ideas for Future Experiments:
My group and I will be prepared to present our poster!
Lab 13: Polymerase Chain Reactions (PCR), Posters, and Gel Preparation
April 13, 2017
Lab 13: Polymerase Chain Reactions (PCR), Posters, and Gel Preparation
Goals: The purpose of the lab is to prepare PCRs and Gel Molds for DNA sequencing. We will also go over the important information pertaining to the upcoming lab poster final.
Background: Week by week we are following the normal protocol a biologist would take to conduct a DNA sequence using Gel Electrophoresis.
Procedure: PCR Protocol
- Label two tubes, one as an experimental and one as a control.
- In the experimental tube add:
- 12.5 microliters of AmpliTaq Gold 360 Master Mix
- 5 microliters of DNA
- 1 microliter of the 20 micro-molarity primer mix
- 6.5 microliters of water
- Total volume is 25 microliters
- In the control tube add:
- 12.5 microliters AmpliTaq Gold 360 Master Mix
- 1 microliter of the 20 micro-molarity primer mix
- 11.5 microliters of water
- Total volume is 25 microliters
- Mix both samples using the vortex, to ensure all the reagents are at the bottom of the microcentrifuge tube.
- Then place both samples in the thermocycler to conduct a thermal cycling profile:
- Initial denaturation: 94 degrees Celsius for 2.5 minutes
- 35 cycles of:
- Denaturation: 94 degrees Celsius for 30 seconds
- Primer annealing: 56 degrees Celsius for 20 seconds
- Primer elongation: 72 degrees Celsius for 2.5 minutes
- Extension: 72 degrees Celsius for 5 minutes
- Hold at 4 degrees Celsius
Making Agarose Gel
- Combine agarose and 1xTAE in an Erlenmeyer flask
- Cover and heat the mixture until the solution is clear, typically until the solution begins to boil
- Cool and add ethidium bromide
- While the solution is cooling, set up the gel box. Make sure it does not leak.
- After cooling, pour the agarose into the gel box and fill halfway up the teeth of the comb
- Let it sit for 30 minutes.
- Cover gel with buffer solution (so that it won’t dry), remove the comb.
- Lastly, situate the gel so that the wells are closest to the negative electrode.
Observations:
Setting up a PCR reaction was fun but we had to be extra careful about contamination. We were not supposed to leave the tubes open very long and were required to wear gloves because any contamination could jeopardize the validity of our experiment. I adopted DNA sample PCS4-2.
I enjoyed making the gels for next lab. The hot agarose had a very strong smell to them.
Ideas for Future Experiments:
I will prepare to use the gels next week and continue to explore ciliate literature.
Lab 12: DNA Extraction
April 6, 2017
Lab12: DNA Extraction
Goals: The purpose of this lab is to perform a Modified Chelex Extraction protocol to extract the DNA from the ciliates our cultures to prepare for future DNA sequencing.
Background: The past few labs we were culturing ciliates for DNA extraction and further DNA sequencing. Since I did not have any live cultures, I planned to adopt someone else’s culture today.
Procedures: Modified Chelex Extraction Protocol
- Transfer 300-500 microliters of ciliate culture to a microcentrifuge tube, using a p1000 micro-pipette.
- Label your tube with your identifier.
- Then centrifuge at 6000g for 5 minutes and discard the supernatant.
- Add 200 microliters, using a p200 micro-pipette, of a 5% Chelex solution and vortex for 1 minute.
- Incubate microcentrifuge tubes in a 56 oC water bath for 30 minutes.
- Then boil for 8 minutes in 100 oC water bath.
- Vortex again for 1 minute.
- Centrifuge at 16000g for 3 more minutes.
- Transfer supernatant to a clean microcentrifuge tube and discard Chelex beads.
Results:
Observations:
I did not get to perform this procedure because I was sick. I also could not attend open-lab because of this. Figure 1 shows my temperature at its lowest over the course of four days.
Ideas for Future Experiments:
First, do not get sick!
Second, I will adopt extracted DNA to continue along with the lab.
Lab 11: Ciliate Picking, Culturing, and Characterizing
March 30, 2017
Lab 11: Ciliate Picking, Culturing, and Characterizing
Goals: The purpose of this lab is to continue picking ciliates to create cultures. Those who already have cultures are to try and classify their ciliates.
Background: As a class, we are attempting to culture ciliates and classify them using physical characteristics and DNA sequencing. The pre-lab work gave us enough information to try and classify ciliates based on their physical appearance and some soil metadata.
Procedures: Ciliate Capture and Culture
- Observe your non-flooded plate under the dissecting scope.
- Capture any ciliates using a p10 micro-pipetter at 5 microliters and place in a 24-well plate.
- Add 1,000 microliters of Cerophyll to the well.
Observations:
At the previous open-lab, I captured and cultured 4 similar looking ciliates. Since the ciliates were way too small for me to classify under the dissecting scope, I decided to wait until the cultures grew so that I could take a sample to observe under the compound microscope. However, when I returned this week, my cultures were dead and I did not get to classify my ciliate. To make matters worse, I did not find more ciliates.
Ideas for Future Experiments:
I will continue to try and find more ciliates, but it looks like it is time to adopt a culture.
Lab 10: Metadata and Ciliate Capturing
March 23, 2017
Lab 10: Metadata and Ciliate Capturing
Goals: The purpose of this lab is to obtain the final soil metadata and begin to capture ciliates found in the non-flooded plates while focusing on our growth mindset as scientists.
Background: Soil metadata collection and non-flooded plates were made the previous week for this week’s observation.
Procedures-1: Percent Water Composition
- Obtain soil and weigh-boat from the cabinet.
- Mass it.
- Calculate percent water by subtracting the initial and final mass of the soil and weigh-boat. Then divide the difference by the initial mass and multiply by 100 to get percent water composition.
Procedures-2: Obtain the pH of Soil
- Dip the end of pH paper into the water of your Falcon tube.
- Let sit for 1 minute and record pH.
Procedures-3: Soil Composition
- Measure each layer in your falcon tube to the nearest millimeter and record.
- Take each measurement and divide it by the total length and multiply it by 100 to get the soil composition for each layer.
- Use a Soil Composition Triangle to determine the type of soil.
Procedures-4: Ciliate Capture
- Observe your non-flooded plate under the dissecting scope.
- Capture any ciliates using a micro-pipetter at 5 microliters and place in a 24-well plate.
- Add 1,000 microliters of Cerophyll to the well.
Results:
Observations:
- 7.2 grams – 6.5 grams = 0.7/7.2 = 0.097 * 100 = 9.7% water
- Figure 1 shows pH paper reads 7.5 soil pH.
- Soil was Sandy Clay Loam
- 72% sand
- 25% clay
- 3% silt
- Figure 3 is the chart we used to decide what our soil was.
- Figure 4 demonstrates how I felt after observing my non-flooded plate this time around. No, ciliates, just nematodes! Although, I did research and learn that nematodes and protozoa compete for food sources, so if you have more of one, you have less of the other. I made another non-flooded plate, using the same soil to see if I yield different results.
Ideas for the Future:
I will check on my second soil sample after 24-hours. I aim to successfully culture ciliates from this sample!
Lab 9: Soil Collection and Introductory Metadata
March 16, 2017
Lab 9: Soil Collection and Introductory Metadata
Goals: The purpose of this lab is to learn how to make and use a non-flooded plate to collect soil metadata and ciliates for culturing.
Background: Soil samples were collected over spring break, prior to the lab and we were asked to record several descriptions of the soil and weather at the time of day it was collected. For example, we recorded location, percent humidity, and temperature.
Procedures-1: Soil Collection
- Collect approximately 1 cup of soil from any legal, nonhazardous area to bring to the next lab. Make sure you collect soil no further than 3-4cm deep.
- Ensure you collect appropriate metadata for the outside-class section.
Procedures-2 (a): Soil Metadata: Water Content
- Weigh the weigh boat.
- Add approximately 5 grams of soil and record mass.
- Label with your identifier and place in the cabinet to sit for a week.
Procedures-2 (b): Soil Metadata: Soil Composition
- Label 1 Falcon tube with your soil identifier.
- Add soil to the 5-milliliter mark.
- Add water to the 10-milliter mark.
- Place in Falcon tube stand for next week.
Procedures-3: Setting Up a Non-flooded Plate
- Put 10-15g of fresh or air-dried soil in a Petri dish.
- Saturate with approximately 5-20 milliliters of water but do not flood.
- Observe your soil using the dissecting microscope and record your observations.
Observations:
- I collected my soil outside of the Baylor Sciences Building in an area of feild where the grass was not growing. The weather was overcast with a temperature of 19 degrees celsius, and a humidity of 76%. The last time it had rained was MArch 13, 2017, three days before I collected the soil. Another note is that this area tends to collect water until it is one large puddle. No bushes or trees were within a three-meter radius of the area.
- Procedures-2 (A):
- The weigh boat weighed: 2.2 grams
- Soil weighed: 5.0 grams
- Total weight: 7.2 grams
- Identifier = KAC110502SP17
- The soil in the Falcon Tube began to settle nicely. The forming layers of soil will be very easy to distinguish.
- In my non-flooded plate, I did not see any ciliates, however, I did find a single nematode.
Ideas for Future Experiments:
I will check on my metadata in 24 hours and observe my non-flooded plate again to see if any ciliates emerge. Hopefully, I will find some to culture!
