Date: May 1, 2017

Classifying Ciliates using DNA Extraction

FIGURE 1:

In Figure 1 we have our final poster project. This summarizes what we have been doing over the past few weeks, while classifying ciliates.

Procedures:

Soil and Metadata collection:

1. Collected a soil sample from Iowa in a corn field because Iowa has rich soil.
2. Record information about our soil such as, location (GPS), date, time of day, temperature, last rainfall, humidity, color, etc..
   • Location/Climate
     • Iowan Cornfield
     • Latitude: 41.470282
     • Longitude: -95.318582
     • Date: March 11, 2017
     • Time: 6:30 pm
     • Temperature: 29 degrees F
     • Humidity: 65%
     • Last Rainfall: 1 month prior
     • Color: Brown
3.  Then transferred 5 g of the sample into a weighing boat, and weighed the new sample.
   1. Wait 1 week and weigh the boat again.
   2. Calculate the percent composition of the sand, silt, and clay.
      1. % Sand –> 0%
      2. % Clay –> 5.41%
      3. % Silt –> 94.59%
   3. Obtain the pH of the dirt with pH paper
      1. Ours was pH was 6.5 = acidic
4. Added soil to 5mL mark of the falcon tube and added water until the 10mL mark. Then I vortexed the mixture.
   1. This is to help find what type of soil it is.
5. After a week, the falcon tube divided the mixture into layers.
   1. Measure the layers using a ruler and do some calculations to get a percent composition.
   2. Our dirt was mainly silt.
6. Next I took the soil sample and placed it into a non-flooded dish, and put just enough water to where there is some runoff, but didn’t flooding it. Then observed under a microscope.
   1. After three days of searching, I found no sign of ciliate life.

DNA Extraction:

1. I borrowed my new sample from the infamous Lily because she knew how to raise her ciliates.
   1. Her % composition was:
      • Sand–>92%
      • Silt–>0.4%
      • Clay–>8%
2. Transfered 400uL of her cultured ciliates into a centrifuge tube.
3. Centrifuged the tube for 5 minutes at 6000g.
4. I Added 200uL of Chelex into the tube and vortexed for 1 minute
5. Incubate at 56 degrees C for 8 minutes
6. Then place the tube in a 100 degrees C water bath for 8 minutes.
7. Centrifuge at 16000g for 3 minutes.
8. Transfer the supernatant into another centrifuge tube and label with the Soil identifier
   1. HHN0617
9. Then to determine the purity of the sample, a nanodrop spectrometer was used.

PCR:

1. 12.5 uL 2x Master Mis, 5uL DNA, 1uL of uM primer and water was added to form a PCR mixture.
   • It was added to the 25uL volume mark.
2. The control tube didn’t have DNA but had more water, while the experimental tube had the more DNA
   1. Label the control tube 6OC and the experimental tube 6O.
3. Both of the tubes were vortexed for 10 seconds and stored until the lab was over.

Gel Electrophoresis:

1. We added 100 ml of 1XTAE and 1.5 g of argose to a 250 mL flask and microwaved.
2. We assembled the tray by placing the comb into the tray with the two bumpers on each side.
3. We filled the tray with gel about halfway up the combs.
4. Thermocycling procedure:
   1. Denaturation at 95 degrees C for 30s
   2. 35 cycles:
      1. Denaturation at 95 degrees C for 30s
      2. Annealing at 56 degrees for 20 s
      3. Elongation at 72 degrees C for 2:30s
   3. Extension at 72 degrees C for 5 minutes
5. Added 5 ul of the ladder to the product using a micropipet into the gel tray.
6. We then placed the lid on the tray after loading the DNA samples into their respected loading zones, and turned it on at 100 volts for 30 minutes
7. We placed the tray under UV light so we could actually see the results.

Here is a visual representation of our Gel Electrophoresis:

Labeling Info:

• Soil Identifier: HHN0617
• PCR Label: 6O

Ideas for Future Experiments:

• Make sure to double check your work, even if you think you did it right the first time.
• Don’t be afraid to question the results or the process
• Use more than one sample

Date: April 20, 2017

Objective:

The objective of this lab was for us to understand the process of Gel Electrophoresis and for us to continue our poster designs.

Background:

In the last lab we completed PCR (Polymerase Chain Reaction) which leads us into Gel Electophoresis.

Procedures:

• We had to repeat our PCR due to some technical difficulties, so follow the procedures form the last lab notebook.

Loading the Gels

• Using DNA from the last lab that was contaminated, we practiced loading the wells for Gel Electrophoriesis.
  • Extract 10 uL DNA with pipette and with a steady hand carefully load the DNA into the gel wells.
  • Watch as the gel box  sent volts of electricity through the gel. This shows us the size of the DNA we are observing.

Posters

• Traveled to the computer lab for us to start on the process of our poster designs.
• We collaborated with each other on where we wanted to start, then met up outside of lab to finish the poster.

Observations/Results: 

We got to watch electric currents shoot through our Gel, so we could see the different DNA sizes.

Ideas for Future Experiments:

Come prepared to the last lab, which is our final presentations.

Date: March 16, 2017

Learning Objectives:

• In this we will continue to learn how to set up a non-flooded plate
• Take samples of soil from various locations during Spring Break (Mine –> Iowa)
  • Be able to describe the % of sand, silt, and clay in your collection
• How to culture and observe soil ciliates.

Background:

In the previous labs, we have been observing different samples of soils, so now we are using the soil from a different location to see the if there are any differences in the ciliates compared to the previous observations.

Procedure:

Soil Metadata –> The soil collected was from the GREAT state of Iowa.

• Weigh a weigh boat = 2.2g
• Add ~5.0 grams soil to the weigh boat
  • Ours was 5.2 grams.
  • Total weight = 7.4 grams
• Place the soil sample in a petri dish
  • Saturate the soil, but don’t flood the sample.
  • Observe under a dissecting microscope.

Falcon Tube Protocol 

• Label the tube (HHN06S17)
• Add 5mL of soil to the falcon tube
• Fill the tube to 10mL with distilled water
• Add 1 drop of resuspension solution
  • Mix using the vortex
• Let sit to allow the solution to separate.

• Observe more from the from the petri dish
• Separate the top liquid layer.

Results/Data:

Observations:

• Not much movement
•  1 ciliate found
  • Color: Clear
• Swimming too quickly to possibly see.

Ideas for Future Experiments:

• Try not to over-saturate the soil
• Don’t give up looking at the soil, even when you don’t find anything the first few times around.
• Check after a period of time, to check for life.

Date: March 2, 2017

Learning Objectives:

• In this lab we set out to learn more about the scientific thinking for our Lab Reports, while being able to peer review another paper, and use the critiques on our papers to better them.
• We are also going to observe soil Ciliates in non-flooded plates.

Background:

• In past labs, we have been observing the different ciliates under microscopes, so this is nothing new. Now we are looking a little deeper to determine the ciliate classification.

Procedure:

Lab Reports

• Review another students lab report that had a different concentration than yours, so that we can see the different results. (.0025%)
  • Give positive feedback and make sure that you aren’t just telling them what to do, but suggesting either a better way or a more efficient solution.

Soil Ciliates

• Add 10-50 g of your soil into a petri dish
• Add distilled water to saturate the dish, but don’t flood the petri dish.
• Observe the petri dish with soil sample under a dissecting microscope and record your observations.
• Transfer 100 microliters of the liquid soil sample using the 20-200 micropipette to a small watch class to observe under a compound microscope.
• Observe and record your observations.

Results/Data:

For our peer review, we received our Lab Reports back and make corrections as needed.

For our soil ciliates, we could not find any living ciliates in the soil we observed after a few trial, all we found were dead ciliates.

Ideas for Future Experiments:

For the reports, we should make sure that the rough draft is complete, so that it helps not only you as the writer, but the reviewer be able to make full corrections.

For the soil ciliates, to not over saturate our samples.

February 26, 2017

Lab 7 – Data Analysis

Learning Objective:

For this lab our objectives were to, compile the results of every partner in our group and present them to the class, be able to understand the criticism and use that to our advantage, and use that criticism to better our graphs and charts.

Background:

From the previous labs we have been collecting hard counts of the number of cells in a certain sample of a controlled group and a treatment group of tetrahymena. The treatment group was treated with a pesticide called gamma cyhaylothrin.  We are going to use our data and results to create a graph of our numbers.

Procedure:

• Collect each partners counts, average those counts, and place them into a graph using Excel.
• Present the graphs to the class, and receive feedback from the professor and teacher assistance’s.

Data:

Ideas for Future Experiments:

Using the comments and feedback we got from class will really help us make adjustments to our graph, so that we are able to present them in a way that is more efficient for everyone.

February 26, 2017

Lab 6 – Cell Counting and Pesticide Treatment Part II

Learning Objectives:

The learning objectives for this lab are to continue to learn how to count cells, review the percent calculations from the data we collect, and use the results from the data to help us write our conclusion portion of our scientific report.

Background:

We have been counting certain samples of cells for a controlled group and a treated group. Last lab we counted the number of tetrahymena treated with a pesticide after waiting 24 hours.

Procedures:

• Stir the well-plate
• Take 2 uL of the 1 week samples of controlled and treated group and place them on a concavity slide to count and observe.
• Make 2 counts for each well.
• To Dilute the substance because there are too many to count, or because the sample was moving to quick, these are the steps:
  • Take 45.0 uL of PPT and place it in a plastic plate.
  • Take 5.0 uL of either the controlled or treatment group and mix the two solutions together
  • Take 2.0 uL of this new sample and place it on a concavity slide to count and observe
    • Multiply the number of cells you counted by the dilution factor.

Observations/Raw Experiment Data:

• Controlled group after 1 week:
  • C4 – 10 cells per 2 uL
  • C5 – 11 cells per 2 uL
  • C6 – 36 cells per 2 uL
• Treatment group after 1 week
  • D4 – 36 cells per 2 uL
  • D5 – 56 cells per 2 uL
  • D6 – 147 cells per 2 uL

As we can see here compared to lab 5 the number of cells per group significantly decreased over time. There was no affect that the pesticide had on the treatment group because both of the numbers from the data, both decreased but the treated group still had more cells in their samples.

Ideas for Future Experiments:

For tests/experiments in the future, we must be sure there is no error when transferring our samples, or when mixing our solutions.

The next part of this experiment is to take our data and be able to represent our data in graphs.