Danielle Eberwein

11/19/16

Objective:

The purpose of this experiment was to learn how to fill the wells in the gel and how to perform Gel Electrophoresis. After imaging it, it will show if the DNA was extracted from our soil samples.

Procedure:

1. The Agarose gel was already prepared for us.
2. Make 1xTAE in 1L Erlenmeyer flask from  the already made stock solution.
3. For 1 Liter of 1xTAE, I needed 20 mL of 50xTAE stock solution and 980 mL of D.I. water.
4. Set up gel electrophoresis box, make sure the open ends are sealed.
5. Make sure the comb is inserted with its back towards the nearest edge.
6. Pour the gel into the prepared mold.
7. Allow to sit for 30 minutes to solidify.
8. Cover gel with the prepared 1xTAE buffer solution.
9. Retrieve the 5 µL ladder, 10 µL buffer and the 4 PCR tubes with your letters on it from the back.
10. Add the buffer to any of the 4 PCR tubes that do not show color.
11. Micropipette 5 uL of the ladder into well 1.
12. Micropipette 10 uL of the PCR sample into wells 2-5 in the box.
13. After this is done, place the lid on your box and turn on the power supply to 100 volts.
14. Allow to run for around 30 minutes, or around halfway.
15. Image with UV light in the lab upstairs to get the results of the DNA.

Results:

1.DNA Ladder

2.D1

3.D2

4.D3

5.D4

6.DNA Ladder

7.F4

8.F3

9.F2

10.F1

Our sample was inconclusive, which might of had to do with the crack on the edge of the gel. There were no DNA bands visible for our sample.

Conclusion:

The gel was disposed of in the appropriate safety bag and Josie stored our remaining samples.

Danielle Eberwein

11/15/2016

Objective:

The purpose of this experiment was to prepare for our poster project and to prepare the DNA samples made in the last lab for PCR. There were 4 tubes made, 2 were controls and 2 had soil DNA.

Procedure:

1. Prepare PCR in the total volume of 25 uL of reaction mixture.
2. Fill out the table to see how much DNA and Water is needed for the EUK and COX primers.
3. There is a constant of 12.5 uL of AmpliTaq Gold 360 2X master mix for all 4 tubes.
4. There can be a range from 2.5-10 uL of DNA depending on your DNA sample.
5. Calculate the volume of your DNA that equals about 100 ng.
6. Add 1.25 uL of the 20 uM primer to the correct tubes.
7. Calculate the amount of water that is required for each tube to be equal to 25 uL. This depends on the volume of DNA for the EUK and COX1.
8. There is a control tube for each reactant, which will contain extra water instead of DNA.
9. Vortex all the samples, to mix.
10. Use the thermal cycling profile.
11. Initial denaturation is 94°C for 2.5 minutes.
12. 35 cycles:Denaturation: 95°Cfor 30 seconds

    Primer annealing: 58°C for 1 minute

    Primer elongation: 65°C for 2.5 minutes

    Extension: 72°C for 8 minutes

Results:

Conclusion:

Next week we will look at the DNA again after it has denatured and perform Gel electro phoresis. My PCR label is F. The tubes are labeled F1, F2, F3, F4.

Danielle Eberwein

11/8/2016

Objective:

The purpose of this experiment was to extract DNA from the ciliates that we cultured, but since I did not have any ciliates I used my soil and the MoBIO method to extract DNA from my soil.

Procedure:

• Use the PowerBead Tubes that are provided and add 0.25 g of your soil sample.
• Gently vortex the tube to mix.
• Heat Solution C1 to 60 degrees until dissolved.
• Add 60 uL of Solution C1 and vortex it briefly.
• Make sure the PowerBead tubes are secured in the horizontal vortex adapter and vortex at maximum speed for 10 minutes.
• Make sure there is no rubbing of the tubes. Centrifuge the tubes at 10,000 x g for 30 seconds at room temperature. Make sure not to exceed this limit.
• Transfer this to a 2 mL collection tube provided.
• Add 250 uL of Solution C2 and vortex for 5 seconds to mix.
• Incubate this at 4 degrees in the freezer C for 5 minutes.
• Centrifuge the tubes for 1 minute at 10,000 x g at room temperature.
• Transfer up to 600 uL of supernatant to a clean 2 mL collection tube that is provided.
• Add 200 uL of Solution C3 and vortex for a couple seconds. Incubate it again in the freezer at  degrees C for 5 minutes.
• Centrifuge the tubes at 10,000 x g for 1 minute at room temperature.
• Now transfer up to 750 uL of supernatant into a clean 2 mL collection tube that is provided.
• Shake Solution C4 before using it and then add 1200 uL of Solution C4 to the supernatant and vortex for 5 seconds.
• Add 675 uL onto a spin filter and centrifuge it at 10,000 x g for 1 minute at room temperature.
• Discard the flow through and add another 675 uL of supernatant to the spin filter and centrifuge it at 10,000 x g for 1 minute at room temperature.
• Then add the remaining amount of supernantant and repeat.
• Add 500 uL of Solution C5 and centrifuge for 30 seconds at 10,000 x g at room temperature.
• Discard the flow through and then centrifuge it again at room temperature for 1 minute at 10,000 x g.
• Place the spin filter into another clean 2 mL collection tube and avoid splashing any of the Solution C5 onto the spin filter.
• Add 100 uL of Solution C6 to the center of the white filter membrane.
• Centrifuge this for 30 seconds at 10,000 x g at room temperature.
• Discard the spin filter and the DNA in the tube is now ready.

Observations:

Even though we weren’t able to use our ciliates, we still got to extract DNA from our original soil sample. We ran out of time and stopped at step 17, the remainder steps were finished by our TAs.

Conclusion:

Our tube is labeled JD and is in the fridge in the back of the room.

Danielle Eberwein

11/1/16

Objective:

The purpose of this experiment was to look at the ciliates from last week and examine them. We also need to relook at the tubes that were made in the past experiments to determine the composition of the soil (silt or clay).

Procedure:

• Take out your stereoscope microscope, 24-well plate, soil samples and Falcon tube.
• Look for ciliate culture in your well plate.
• If there are any ciliates present, take a picture and dye it.
• If there are none, look in your soil sample or adopt from another person’s soil.
• Take the Falcon tube and measure the percent of clay, sand and silt in it.

Observations:

There was a very large population in all 3 of my well plates. The ciliates were very small, so there might be a chance of contamination from Tetrahymena. I did not have time to take pictures of my ciliates, but next week I am going to try to obtain another ciliate from my soil sample to see how it differs.

Results:

My Falcon tube contained .5 mL clay and .5 mL of silt.

Danielle Eberwein

10/25/16

Objective:

The purpose of this experiment was to look for ciliates in the soil sample from the previous lab and to transfer them to our 24-well plates. We also were supposed to calculate and reflect on geologic time. We looked at how evolution occurs in ciliates by talking about natural selection, sexual selection, genetic drift, gene flow and mutation.

Procedure:

• Obtain stereoscope microscope, 24-well plate and both of the soil samples with your name on them in the back of the room.
• Look in the non-flooded plate and search for ciliates.
• Once you find a ciliate, take a 10 uL pipette and suck the ciliate out of the soil sample. Place it on the lid of the petri dish.
• Add a drop of Cerophyll to the ciliate and observe under microscope to make sure you obtained it.
• Once you see you have it, add 500 uL of Cerophyll to one of the well plates and transfer the ciliate to the same well plate.
• Repeat this until you have a couple of ciliates in multiple well plates.
• If you cannot find any ciliates in your soil sample, adopt from another person’s.

Observations:

The ciliates were a lot easier to find this week, probably because they got water added to them. Last week I couldn’t find any ciliates in my non-flooded plate, but I found a lot in it this week. It was hard to just capture one on its own, so there is multiple in each of the well plates.

Conclusion:

The ciliates are supposed to duplicate every 70 hours, so next week there should be a lot more ciliates in my well plate. My 24-well plate and both soil samples are in the back drawer labeled DLE09F16.

Danielle Eberwein

10/18/16

Purpose:

The purpose of this experiment is to find the soil samples % water content and % of each soil type of soil particle. The non-flooded plate was looked at to observe the ciliate content. We are looking at how they are classified and their evolution

Procedure:

• Take out your dissecting microscope and get your soil samples from the back of the room.
• Take your dry soil sample and calculate the % water content.
• Take 2 Falcon tubes and add 5ml of soil without grass or large clods in it. Add water to the 10ml mark. Add a drop of the resuspension solution and shake this solution for around a minute.
• Let the tube stand for one minute and let the sand settle at the bottom.
• Pour the suspension into the second tube.
• Record the mass of the sand in the first tube.
• Write your name on the tubes and store them together till next week’s experiment.
• The silt and clay well settle out and the % of each will be measured.
• Next, examine the non-flooded plate and look in the areas with the most water.
• Look for ciliates in the soil water surface and remove it using 2o µm using a 20 µm
• Transfer the ciliate to the lid of your petri dish.
• Add a drop of Cerophyll using a 10 µm pipette to a different spot on the petri dish lid.
• Transfer the ciliate from the first drop to the drop of Cerophyll to isolate it from the other organisms.
• Take your 24 well plate from the back of the room and clean it out using alcohol and water.
• Transfer this isolated ciliate to your 24 well plate.
• Add 500 µm of Cerophyll into the same well plate as the isolated ciliate.
• Store the 24 well plate in the back of the room with your name on it.

Observations:

I could not find any ciliates in my soil sample. After looking for a long time, I found a ciliate in Taylor Guynup’s soil sample. After transferring it to the 24 well plate, the ciliate was lost. It was very hard to try to capture the ciliate because they move so fast and trying to transfer it 2 times made it even easier to lose it.

Conclusion:

My 24-well plate and my 2 petri dishes are in the back of the room with my name on it. The 2 tubes are in the rack at the front with everyone else, labeled DLE09F16.

Danielle Eberwein

10/11/16

Purpose:

The purpose of this experiment is to look at soil biodiversity and its function. We are going to look at soil water content, non-flooded plates, soil pH, ion content and ciliate abundance.

Procedure:

1. Label 2 petri dishes
2. Measure the empty petri dish with the lid on it.
3. Measure the plate with soil in it and lid on it as well.
4. Record the masses of both of these and set the water content plate aside.
5. Add water to your non flooded plate and leave it on desk to observe later.
6. Obtain a microfuge tube and weigh and record it.
7. Add soil to the 250uL mark and measure the weight and record.
8. Add up to 1000uL with DI water to the microfuge tube.
9. Vortex it for a couple of seconds to mix it.
10. Let the soil settle
11. Measure the pH of the soil with a pH strip.
12. Obtain the compound microscope, slide, slid cover and 1-10uL pipette.
13. Put 1 uL of the non-flooded plate soil on the slid.
14. Put Vaseline on all 4 corners of the slide cover and carefully place it on top of slide with soil.
15. Put this slide under the compound microscope and observe what organisms are present. Record this data.

Observations:

From the soil 10ft away from the creek I found ciliates, rotifers and nematodes. My soil contained a lot of grass and was clumped together, so it was pretty hard to see organisms in it.

Conclusion:

Observing the soil from different places along the creek contributed to different organisms being found in the soil. My partner only found ciliates in his soil, while I had a diversity of organisms. Also, my soil was already moist while his was more dry.

Danielle Eberwein

10/4/16

Purpose:

The purpose of this experiment was to go over everything that we did leading up todays experiment. The lab techniques that we learned about were microscopes, staining, measuring volumes, making dilutions, culture techniques and cell counting. We got our experiments out and observed them to get our final data before we have to turn in our lab report.

Procedure:

1. Obtain the 24 well-plate from the back of the room with your Tetrahymena in it.
2. Use the stereoscope microscope in this experiment.
3. Obtain a watch glass.
4. Get a 1-10 uL pipette, and a 100-100 uL pipette.
5. Get the tips for the pipettes and Tetrahymena medium from the front.
6. Obtain all 3 24-well plates with the 3 different volumes in them.
7. Create a serial dilution of 1:10, by adding 1 uL of the first well, and 9 uL of the Tetrahymena medium.
8. Observe under the microscope and record the number of living Tetrahymena.
9. Repeat these steps 2 more times for well 2 and 3.

Observations:

500uL (100uL Tetrahymena and 400uL Peptone)

1000uL (100uL Tetrahymena and 900uL Peptone)

1500uL (100uL Tetrahymena and 1400uL Peptone)

Conclusion:

The results of the experiment were flawed because of the bacteria grown in the 1000uL and 1500uL volumes. The original hypothesis turns out not to be correct after the week 2 trial.

We also talked about the upcoming lab 8 experiment. We have to obtain soil from the Baylor creek behind the BSB. If you are an even number you take soil from 10ft away from the creek and odd numbers take it 1ft from the edge.

Danielle Eberwein

9/27/16

Purpose:

The purpose of this lab was to distinguish between valid primary literature and other online sources, by practicing reading journal articles. Also to review the calculations of % solutions and perform easy serial dilutions in a watch glass. We will also use Methyl green-pyronin stain to observe culture. Last, we will set up our experiment or measure results.

Procedure:

1. Obtain both the compound microscope and stereoscope microscope.
2. Gently clean the microscope lenses.
3. Obtain a slide and clean it properly with alcohol.
4. Put a single drop of the Tetrahymena culture on the slide.
5. Obtain a cover slip and vaseline.
6. Apply the vaseline to the corners of the coverslip to keep the slide from crushing the Tetrahymena.
7. Use the coverslip and place it over the tetrahymena culture.
8. Add a drop of methyl green-pyronin stain to the edge of the coverslip and let it disperse into the Tetrahymena culture.
9. Put the slide under the compound microscope and observe.

Observation:

By adding the methyl green-pyronin stain to the tetrahymena, it made it a lot easier to locate where the Tetrahymena were on the slide. I could clearly see the shape and outline of the Tetrahymena and their two nuclei in the cell.

Conclusion: Staining cell cultures is a really good way to see the cell more vividly under the microscope. It gives more definition of the cell and makes it easier to locate them.

Experiment results:

Purpose:

We are going to observe and record the number of Tetrahymena in the different volumes of Peptone after 5 days.

Procedure:

1. Use the stereoscope microscope in this experiment.
2. Obtain a watch glass.
3. Get a 1-10 uL pipette, and a 100-100 uL pipette.
4. Get the tips for the pipettes and Tetrahymena medium from the front.
5. Obtain all 3 24-well plates with the 3 different volumes in them.
6. Create a serial dilution of 1:10, by adding 1 uL of the first well, and 9 uL of the Tetrahymena medium.
7. Observe under the microscope and record the number of living Tetrahymena.
8. Repeat these steps 2 more times for well 2 and 3, and the control group.

Observations:

There is a bacteria that has grown in all of the Tetrahymena/peptone well plates.

Week 1 Trial:

Conclusion:

Our hypothesis is verifying itself. The Tetrahymena exposed to a higher volume of peptone have more Tetrahymena in them when we observed them after the 5 days.

Danielle Eberwein

9/20/16

Purpose:

Use the compound microscope to study ciliates. Set up our experiment to start on. We learned about compound and stereoscope microscopes, and the difference between them. Stereoscope microscopes is 3 dimensional and has a long working distance, no zoom magnification and has a low magnification. Compound microscope is 2 dimensional, high magnifications, low working distance, and no zoom magnification.

Procedure:

Retrieve both the compound and stereoscope microscopes and take them back to your desk. Clean Microscopes with alcohol and sheets.

Use the prepared slides on the table to look at under the compound microscope.

Focus it on the 4x objective lens, then the 10x, and finish with the 40x.

Put up the prepared slide, and get a blank slide and a coverslip.

Go to the front and put a drop of one of the ciliate components onto your slide and put a sheet over it.

Put it under the compound microscope and observe.

Focus it on the 4x objective lens, then the 10x, and finish with the 40x.

Observations:

I could not see any of the culture from the unknown solution on the slide sheet.

Procedure:

Retrieve your 24 well plate from the week before and dispose of the ciliate cultures properly. Rinse your well plate, with water and bleach.

Now take the 30ml of media and add 180 microliters to 5 clean well plates.

Then take the 2ml Tetrahymena culture given to each group and using a serological pipette, add 20 microliters to the first well plate.

Now dilute the well plate each time by taking 20 microliters from the first well plate and add it to the second, then 20 microliters from the second and add to the third, etc. Dilute by 1:10.

Observations:

There were around 50 Tetrahymena in the first dilute and about 15 in the second. So by multiplying the 15 by 100(since it’s the second dilution, 10×10), there were about 1,500 in the original Tetrahymena culture.

Conclusion:

Serial dilution is very useful in trying to figure out large quantities of a culture. It is very helpful to get practice with this process, since I am using It in my experiment. The compound microscope is a lot more useful in this situation because of the large working space. My well plate is placed in the back of the room in the first drawer, with my name on it.