Purpose: the purpose of this lab was to assemble our posters and prepare for the symposium.

Results/poster:

Abstract:

In current times, there is no working protocol for effectively extracting and characterizing ciliate diversity in soil samples. This work was centered around developing a working protocol for achieving this result. Various methods were used including PowerSoil and Chelex methods of extracting DNA from soil samples. With that, COX-1 and V4 primers in PCR were used to amplify this genetic material. Due to PowerSoil’s many false negatives and Chelex’s high accuracy, it was decided that Chelex was the most effective. It was also decided that V4 was more reliable for the targeted ciliate microorganisms. Gel electrophoresis was another tool utilized with fluorescent staining techniques to analyze the DNA content of samples collected. The decided procedure allows for clean, effective, and reliable results that can be used by other scientists to identify various species of ciliates.

Results:

We were successful in completing the poster an now await critiquing from the professors.

Purpose:

The purpose of this exam was to run the gel elecetrophoresis for the final time using the PCR tubes we made last class and to also brainstorm ideas for our posters for the biology symposium.

Procedure:

1. Place argose 1.8% gel in the Box an cover with buffer just to the point where the gel is slightly covered.
2. Cover the lid to the box matching the colors indicated on both the lid and the gel box.
3. run the gel for 30 minutes
4. Remove the gels using gloves and examine under UV light.
5. Record your findings and begin brainstorming for your groups poster project.

Results:

Our eDNA sample showed promise near the 300-400 band length area.

along with out positive DNA sample that had band around the same area but a little more faint.

Conclusions:

Now that we have completed the analysis we can begin working on our posters which is cumulative of all of the tests and gels we ran through the whole semester.

Purpose:

The purpose of this experiment was to text the new chelex modified protocol we have been perfecting over the semester and run it in a gel so we can determine the most optimum way to determine morphology in Waco.

Process:

Gel electrophoresis set up:

1. Combine and mix 10 mil of the 10x TAE and 90ml of DI water in a flask.
2. Pour until 35 ml remains in the flask.
3. Measure .63g of agarose and add to the flask.
4. place lid over the flask being careful to not place to tight. (make sure the weighing paper is in between the cap and the flask.
5. Microwave at 7 for a minute and 20 seconds.
6. place in cooling bath for 5-6 min and then add 2microliters ethidium bromide and mix gently.
7. Pour the gel into the gel kit making sure there are no bubbles at all and let sit for about 25-30 minutes.
8. Then add a buffer to keep the gel from drying out.
9. Place in refrigerator.

PCR

1. Combine the 5 microliter of master mix and 1 microliter of the eDNA sample in the eDNA sample for the PCR.
2. once complete take to the third floor for incubation.
3. After this, the label your eDNA sample tube and place on rack downstairs.

Conclusion:

We were successful in extracting the DNA sample form the soil sample and were in the process of setting up the gel elctrophoresis. In all, the group was successful in finding samples but we await the results of the lab in order to make out posters.

Purpose:

The purpose of this lab was to determine the results from the last lab by running the gel electrophoresis. After this, we ran the gel under the UV light to determine if we were successful in transferring solid DNA samples to be used in our study.

Procedure:

1. Start by removing the bumper used last class to make the gels.
2. in well one add 10 microliters of positive control Cox1 sample.
3. in well two add 10 microliters of negative control Cox1 sample
4. in well three add 10 microliters of the eDNA Cox1 sample.
5. leave well 4 empty
6. in well five add 10 microliters of the positive control V4
7. in well 6 add 10 microliters of negative control V4
8. in well seven add 10 microliters of eDNA V4
9. in well eight add the ladder
10. Plug in the cords based on color after closing the lid to the gel.
11. run the gel for 30 minutes at 100 volts
12. remove the gel and examine under a UV light.

Results:

The UV examination gave us two promising results:

• A sector of the V4 DNA patch was brightly highlighted and looks very promising.
• The DNA was also slightly present for the Cox1 sample but fainter than the V4 DNA sample.

Conclusion:

We were successful in running the DNA test using the gel electrophoresis which gives insight into our study. However we are still unsure if the DNA acquired is viable for use to determine anything about morphology.

The Purpose:

The Purpose of this experiment was to set up our PCR tubes and our Gel electrophoresis plates so that we can analyze the DNA samples next class.

Procedure:

Using c1v1=c2v2 calculate the proper ratios of concentrate and DI water needed for the tubes.

PCR:

1. Start with 6 microfuge tubes that contain 12.5 microliters of either cox 1 or V4
2. Label the tubes with your initials and sperate the tubes in half leaving 1-3 for Cox 1 and 4-6 are for V4 primers.
3. add 11.4 microliters of water to the negative controls of the two treatments then add 10.9 microliters to the rest.
4. add .6 of Cox1 and V4 to their respective treatment groups.
5. store and record.

Gel electrophoresis set up:

1. Combine and mix 10 mil of the 10x TAE and 90ml of DI water in a flask.
2. Pour until 35 ml remains in the flask.
3. Measure .63g of agarose and add to the flask.
4. place lid over the flask being careful to not place to tight. (make sure the weighing paper is in between the cap and the flask.
5. Microwave at 7 for a minute and 20 seconds.
6. place in cooling bath for 5-6 min and then add 2microliters ethidium bromide and mix gently.
7. Pour the gel into the gel kit making sure there are no bubbles at all and let sit for about 25-30 minutes.
8. Then add a buffer to keep the gel from drying out.
9. Place in refrigerator.

Conclusion:

We were successful in making the agarose gel and finishing the set up for our PCR amplification and therefore can continue or morphology study next class by running the gel eletrophoresis and learning more about the diversity of our habitat.

.

Purpose:

The purpose for this experiment was to have each table in the lab do both the Chelex protocol and the Mo Bio soil protocol. each side f the table would choose the opposite protocol and then compare the protocols in the end to see which was more advantageous to our morphology study.

Procedure (Chelex):

1. Transfer300-500 microliters of dense ciliate culture to a microcentrifuge tube. record the ciliate culture.
2. centrifuge for 5 min at 6000xg
3. Once the tube is labeled, remove the liquid, being careful to not disturb the pellets, and then add another 300-500 microliters to the tube and centrifuge for 5 min at 6000xg.
4. Remove the liquid once again, then add 200 microliters (5% chellex) and vortex for one minute.
5. Once completed, add 15 microliters of the K protienase.
6. for 15 minutes, incubate the tune at 56 degrees Celsius.
7. remove the tube and boil for 8 minutes (100 degrees)
8. vortex for one minute then centrifuge at 16000xg for 3 minutes.
9. Transfer products to a microcentrifuge tube and label.

Procedure (MoBio Power soil):

1. Add a o.25 soil sample to the power bead tubes and vortex.
2. If the solution C1 is not precipitated, then heat at 60 degrees Celsius until the C1 is dissolved.
3. Add 60 microliters of the C1 solution then vortex for an instant.
4. Use the vortex adapters, clap the tube, and then vortex for 10 minutes at the maximum setting.
5. then cetrifuge at 10,000xg for half a minute at room temperature.
6. Place the product in a 2ml collection tube. and add 250 microliters of Of the C2 solution.
7. Vortex for 5 minutes then incubate for 5 minutes at 4 degrees Celsius.
8. then centrifuge at room temperature for one minute at 10000xg then place 750 microliters of the supernant to a 2ml collection tube.
9. add 1200 microliters of the C4 solution to the tube (making sure to shake before use) and vortex for only 5 seconds.
10. Place 675 microliters onto a  spin filter then centrifuge at 10000xg for 1 minute.
11. repeat the process after removing the liquid from the filter after the last tube.
12. place the supernant on a spin filter and centrifuge at 10000xg for a mintue.
13. 500 microliters of the C5 solution will then be added, centrifuge at 10000xg for 30 seconds.
14. discard any liquid that flowed through the filter and centrifuge again at 10000xg for a minute
15. In a 2ml collection tube, place a spin filter and 100 microliters of the solution C6to the centure of the filter and centrifuge for 30 seconds at 10000xg.
16. Store DNA after discarding the filter.

Conclusion/Results:

The experiment was successful in removing DNA samples. While the Chelex pellet protocol uses a more refined sample the Mo Bio protocol is more suitable to the “dirty” or fresh sample. the Mo Bio will yield a more refined result however, due to the amount of centrifugation and solutions. In the end, the protocols for both yielded results and we will Initiate our PCR technique the next lab in order to analyze the DNA for advancements in Ciliate morphology.

Purpose:

The purpose of this lab was to finally run the Gel Electrophoresis after the set up procedures we did last lab. We were hopeful to achieve results and be able to analyse the DNA.

Procedures: (*USE GLOVES*)

1. set up your buffer by adding 30ml of TAE to the flask and then adding 270 ml of DI water. (this will be used to pour over the gel.
2. Picking off from the procedure form last lab, remove the comb from the gel and place into the Electrophoresis container with the well side facing the black.  (“run to red”)
3. Make a dye solution using 5ml of dye and 15 ml of water to practice pipetting into the wells of the gel.
4. place 5ml of dye into each sample tube and mix.
5. Pipette each of your samples and 5ml of the ladder into their own individual wells.
6. run the elctrophoresis for around 40 min.
7. remove the lid and the gel.
8. record your results.

Conclusion:

We were successful in establishing and running our gel elctrophoresis and we now await the results so we can analyse our DNA samples and proceed with our morphology study.

Purpose:

The purpose of this experiment was to set up the Agaros Gel for the Gel Electrophoresis that will take place between the next two labs. We also labeled and set up our DNA with nucleotides in order to amplify our controls and variables.

Procedure:

This is a chart of the Tube setup Process for the DNA Amplification.

Agarose Gel setup:

1. Add 40 ml of 1xTAE to .6g of Agarose in an Erlenmeyer flask.
2. Cover with a weighing paper and loosely fit cap on top to allow for gas exchange.
3. Heat until it is clear and small bubbles form on the bottom when stirred
4. Allow to cool for 5-6 min.
5. Add 2 micro-liters of ethidium bromide then swirl gently.
6. Set up the box needed or the electophoresis, sealing the open ends.
7. pour the Agarose gel into the mold leaving no room for bubbles then place the comb on the very far end of the box.
8. Allow to sit for 25-30 min then pour 1xTAE buffer solution so the gel doesn’t dry.

Conclusion:

We were successful in amplifying or DNA with nucleotides and also being able to make the Agarose gel for the gel electrophoresis that will happen next class.

Purpose:

The purpose of this lab was to evaluate our results form last time and to create a new and effective protocol to amplify our chances of acquiring ciliates in our organic layer.

Procedure:

Add 2.7 ml of soil-jiuce

Add 0.3 ml of stock solution

Place mixture in a small glass bottle

Add 2ml of water without disturbing the ludox

Centrifuge at 4300g for 15 min

draw 2ml of organic layer between the water and the Ludox layer

put organic layer in microfuge tubes and mix well

Take 2ml of the sample per person and observe under a microscope

calculate efficiency- Observes number of cells per ml/ expected number of cells per ml  x  100

Conclusions/ Results:

The concentration of the solute was greater and the possibility of finding ciliates therefore is amplifies through this new procedure and protocol. The mixing and the pi-petting was successful and the weight of the tube was approximately 23.1g.

Purpose:

The purpose of this lab was to conduct the isolation of cell ciliates form a dirt sample using a centrifuge and the Ludox protocol provided by the prelab.

Procedure:

• Prepare the sample by pipetting 8ml of ludox into a 15ml conical tube.
• Once this is done, pipette 2ml of liquid from the soil to the conical tube.
• add a layer of 2ml distilled water to the ludox mixture
• Centrifuge for 15min
• remove a 5ml layer of cells underneath the water layer.
• Transfer to a smaller 15ml comical tube.
• spin in centrifuge once again and dilute to 10ml.
• remove and store in the freezer

Results/conclusion: Once the lab was completed, we now have stable samples for which we can use to conduct our experiments. In our group we only found a couple “possible” ciliates but otherwise the experiment was successful in isolating the cells.