Final Poster

Date of Experiment: 19 April 2018

Experiment Title: Creation of a Poster

Rationale/ Purpose: The goal of this lab is to create a rough draft of the poster and submit for feedback. Additionally, we were to revise our abstract.

Procedure: We went to the computer lab and created a poster on PowerPoint on what we have done this semester.

Observation/Data/Results: Although our presentation is not complete, we have started working on it and will continue to work on it until the final poster is due.

Interpretation/Conclusion/ Next Steps: The next step is to continue to edit the poster and prepare to present it. I will be unable to go to open lab, but my partners will be able to, so with the feedback given from Dr. Adair and others, we will update the poster.

Date of Experiment: 12 April 2018

Experiment Title:Gel Electrophoresis Part 3

Rationale/ Purpose:  The goal of this experiment is to place the samples created in the previous lab into the gel and run the gel. Additionally, we learned about and wrote an abstract for our poseter while the gel was running. After the gel ran for around 30 minutes, we viewed the gel under ultraviolet light.

Procedure: Place the gel created from the previous lab into the box and insert the samples into the wells. The ladder was placed in well 1, – control in well 6, + control in well 7, and eDNA in well 8. Place the lid on the box and run the gel for around 30 minutes. While the gel was running, we learned about creating a poster, wrote an abstract, and designed a poster.

Observation/Data/Results:

Gel under UV Light

There was no band in the negative control or the positive control. There were faint bands in the environmental DNA.

Interpretation/Conclusion/ Next Steps: In this experiment, we were able to see the DNA travel towards the positive side, creating bands based on the size. Our samples did not run as well as we thought. We were able see the bands in the gel, but they were very faint. Also there were no bands in the positive control. In the next lab, we will continue to work on our poster.

Sample Storage: The PCR tubes were labeled 22-6 -, 22-6 +, 22-6 e. The tubes were returned to the Dr. Adair.

Date of Experiment: 05 April 2018

Experiment Title: PCR and Agarose Gel Part 3

Rationale/ Purpose:  The goal of this lab is to create a negative and positive control tube along with our soil DNA tube. In this, we will add V4 primer for amplification. Additionally, we will prepare an agarose gel with wells to place the DNA solution in.

Procedure:

Take 1.5 mL of the liquid from the non flooded plate. Spin at 6,000 g for 5 minutes and remove supernatant. Repeat this step. Add 200 uL of 5% Chelex to pellet and vortex for 1 minute. Add 15 uL of Proteinase K. Incubate in 55.7 C heat block for 30 minutes. Place in 100 C heat block for 8 minutes. Vortex for 1 minute and centrifuge at 16,000 g for 3 minutes. Transfer 100 ul of supernatant from each of the group’s tubes to a clean tube and label.

Add 12.5 uL of 2X Master mix, 1.25 uL of V4 primer, and 11.25 uL of deionized water into a tube. Label this 22-6 -. Add 12.5 uL of 2X Master mix, 1 uL of the positive DNA template (V4+), 1.25 uL of V4 primer, and 10.25 uL of deionized water into another tube. Label this tube 22-6 +. Add 12.5 uL of 2X Master mix, 1 uL of the environmental DNA template, 1.25 uL of V4 primer, and 10.25 uL of deionized water into another tube. Label this tube 22-6 e.

Add 0.6 g of agarose into an Erlenmeyer flask. Pour 35 mL of 1xTAE gel buffer. Cover the opening with weighing paper and place the cap on top. Microwave the solution for 1 minute 20 seconds and then place it into a water bath to cool. While the solution is cooling, prepare the gel electrophoresis box. Seal the open ends. Add 2 uL of ethidium bromide and pour the agarose gel into the box. Place the comb in the solution and let it solidify.

Observation/Data/Results: The concentration of DNA on our sample was 56.6 ng/uL. No water was added to the sample.

Interpretation/Conclusion/ Next Steps:  DNA was extracted using the Chelex procedure. In the lab, we calculated the amounts of each solution to put in the tubes and the purpose of each of the solutions. Additionally, this lab prepared us for the gel electrophoresis for next lab. In the next lab, we will place the DNA solutions we created into the wells of our gel and run electric currents through the gel to create bands in the gel. Then, we will view the bands.

Sample Storage:  The DNA tube was labeled LBP 22-6 chDNA 4-5-18. The PCR tubes were labeled 22-6 -, 22-6 +, 22-6 e.

Date of Experiment: 29 March 2018

Experiment Title: Gel Electrophoresis Part 2

Rationale/ Purpose: The goal of this experiment is to place the samples created in the previous lab into the gel and run the gel. Additionally, we learned about Next Generation Sequencing while the gel was running. After the gel ran for around 30 minutes, we viewed the gel under ultraviolet light.

Procedure: Place the gel created from the previous lab into the box and insert the samples into the wells. The ladder was placed in well 1, – COX1 in well 2, + COX1 in well 3, eDNA COX1 in well 4, -V4 in well 5, +V4 in well 6, and eDNA V4 in well 5. Place the lid on the box and run the gel for around 30 minutes. We ended up running it four 35 minutes. While waiting for gel, we learned about Next Generation Sequencing. When the gel finished running, went to view our gels under ultraviolet light.

Observation/Data/Results:

Gel after running for 35 minutes

Gel under UV light

Interpretation/Conclusion/ Next Steps: In this experiment, we were able to see the DNA travel towards the positive side, creating bands based on the size. Our samples did not run as well as we though. We were not able see the bands in the gel. Maybe there was too much DNA.

Sample Storage: We gave the gel and the sample tubes to Dr. Adair.

Date of Experiment: 22 March 2018

Experiment Title: PCR and Agarose Gel Part 2

Rationale/ Purpose: The goal of this lab is to create a negative and positive control tube along with our soil DNA tube from the previous experiment. In these controls, we will add COX 1 or V4 primers for amplification. Additionally, we will prepare an agarose gel with wells to place the DNA solution in.

Procedure:

Add 12.5 uL of 2X Master mix, 0.63 uL of COX1 primer, and 11.88 uL of deionized water into a tube. Label this tube 1 and put group name on it. Add 12.5 uL of 2X Master mix, 1 uL of the positive DNA template, 0.63 uL of COX1 primer, and 10.88 uL of deionized water into another tube. Label this tube the 2 and put group name on it. Add 12.5 uL of 2X Master mix, 1 uL of the environmental DNA template, 0.63 uL of COX1 primer, and 10.88 uL of deionized water into another tube. Label this tube 3 and put group name on it.

Add 12.5 uL of 2X Master mix, 0.63 uL of V4 primer, and 11.88 uL of deionized water into a tube. Label this tube 4 and put group name on it. Add 12.5 uL of 2X Master mix, 1 uL of the positive DNA template, 0.63 uL of V4 primer, and 10.88 uL of deionized water into another tube. Label this tube the 5 and put group name on it. Add 12.5 uL of 2X Master mix, 1 uL of the environmental DNA template, 0.63 uL of V4 primer, and 10.88 uL of deionized water into another tube. Label this tube 6 and put group name on it.

Add 90 mL of deionized water and 10 mL of the 10xTAE gel buffer to an Erlenmyer flask. This is the 1xTAE gel buffer. Add 0.6 g of agarose into an Erlenmeyer flask. Pour 35 mL of 1xTAE gel buffer. Cover the opening with weighing paper and place the cap on top. Microwave the solution for 1 minute 20 seconds and then place it into a water bath to cool. While the solution is cooling, prepare the gel electrophoresis box. Seal the open ends. Add 2 uL of ethidium bromide and pour the agarose gel into the box. Place the comb in the solution and let it solidify. After it solidifies, remove the comb and pour 1xTAE gel buffer on top until the gel is covered. Label the box and store it for next lab.

Observation/Data/Results: We used the PowerSoil procedure in the previous lab. In order to calculate the amount of COX1 and V4 primer needed to create a final concentration of 0.5 uM, we used the equation V1C1=V2C2. (V1)(20 uM)=(25uL)(0.5 uM) V1=0.625 (we rounded to 0.63). To find the amount of 10xTAE gel buffer, we multiplied 35 and 1.8, then divided by 100. The answer is 0.63, but the scale only reads up to the tenths place, so we just measured 0.6g.

Interpretation/Conclusion/ Next Steps: In the lab, we calculated the amounts of each solution to put in the tubes and the purpose of each of the solutions. Additionally, this lab prepared us for the gel electrophoresis for next lab. In the next lab, we will place the DNA solutions we created into the wells of our gel and run electric currents through the gel to create bands in the gel. Then, we will view the bands.

Sample Storage:  We labeled the tubes 1-6 and wrote LBP on all of them. We placed them on the green rack and put the rack in the front of the room.

Date of Experiment: 15 March 2018

Experiment Title: Chelex and PowerSoil

Rationale/ Purpose: The goal of experiment was to conduct a procedure that would lead to good results. We would follow two procedures, Chelex and PowerSoil, to extract the DNA.

Procedure:

PowerSoil: Add 0.25 g of soil into the PowerBead Tube. Vortex the tube for around 10 seconds. Add 60 uL of Solution C1 and vortex briefly. Vortex the tube for 10 minutes on a flat bed vortex pad. Centrifuge tube for 30 seconds at 10,000 g. Transfer the supernatant to a collection tube. Add 250 uL of Solution 2 into the supernatant and vortex for 5 seconds. Place the tube into the refrigerator for around 5 minutes. Centrifuge the tube for 1 minute at 10,000 g.Transfer 600 uL of the supernatant to a collection tube. Add 200 uL of Solution C3 and vortex. Place the tube into the refrigerator. Centrifuge the tube for 1 minute at 10,000 g.Transfer 750 uL of the supernatant to a collection tube. Add 1.2 mL of Solution C4 and vortex for 5 seconds. Add 675 uL of the solution onto a Spin filter and centrifuge at 10,000 g for 1 minute. Throw away what is in the tube. Add another 675 uL of the solution onto the Spin filter and centrifuge at 10,000 g for 1 minute. Throw away what is in the tube.Add the remaining solution onto the Spin filter and centrifuge at 10,000 g for 1 minute. Throw away what is in the tube. Add 500 uL of Solution C5 and centrifuge at 10,000 g for 30 seconds. Discard what is in the tube. Centrifuge tube for 1 minute at 10,000 g. Place the Spin Filter into a clean collection tube. Add 100 uL of Solution 6 to the white filter. Centrifuge at 10,000 g for 30 seconds. Discard the Spin Filter. Label the tube and place into refrigerator.

Chelex: Add around 400 uL of dense ciliate culture to a microcentrifuge tube. Centrifuge at 6,000 g for 5 minutes. Throw away supernatant. Weigh 0.5 g Chelex and add to a 15 mL conical tube, Add deionized water up to the 10 mL line. Add 200 uL of 5% Chelex to pellet and vortex for 1 minute. Cut off the tip of a 1000 uL micropipette tip and add 15 uL of proteinase. Incubate in 56 C heat block for 30 minutes. Place in 100 C heat block for 8 minutes. Vortex for 1 minute and centrifuge at 16,000 g for 3 minutes. Transfer supernatant with DNA in solution to a clean tube and label. Take 1.5 mL of the liquid from the non flooded plate. Spin at 6,000 g for 5 minutes and remove supernatant. Repeat this step 2-3 times. Weigh 0.5 g Chelex and add to a 15 mL conical tube, Add deionized water up to the 10 mL line. Add 200 uL of 5% Chelex to pellet and vortex for 1 minute. Cut off the tip of a 1000 uL micropipette tip and add 15 uL of proteinase. Incubate in 56 C heat block for 30 minutes. Place in 100 C heat block for 8 minutes. Vortex for 1 minute and centrifuge at 16,000 g for 3 minutes. Transfer supernatant with DNA in solution to a clean tube and label.

Observation/Data/Results: We did the PowerSoil experiment. The weight of our soil was 0.2.

Interpretation/Conclusion/ Next Steps: We were able to analyze our results and figure out what went wrong in our previous experiments. In the next lab, we will perform PCR and gel electrophoresis to the DNA we isolated.

Sample Storage:  We placed our group name and number ( LBP, 6) along with the procedure we followed (M for MoBIO), into the refrigerator.

Date of Experiment: 01 March 2018

Experiment Title: Gel Electrophoresis

Rationale/ Purpose: The goal of this experiment was to place the DNA sample, controls, and ladder into the wells of the gel from last lab. Additionally, we peer edited introduction paragraphs.

Procedure: Create a buffer by adding 30 mL of TAE buffer and 270 mL of deionized water into an Erlenmeyer flask. Swirl to mix and set aside. Obtain the gel from last lab and place into the gel electrophoresis box with the wells closest to the negative side (black). Pour the buffer on top of the gel.

In a tube, add 5 uL of loading buffer and 25 uL of deionized water. Practice placing solution into the wells three times pipetting 10uL of the dye and water solution. Obtain the tubes from last lab and add 5 uL of loading buffer into each of the tubes. Pipette 5 uL of ladder into a well. Carefully pipette 10 uL of the positive and negative control and soil DNA tube into the wells. Close the lid and plug in the cords. Run the gel electrophoresis at 110V for 30 minutes.

After 30 minutes, take the gel out of the box and store for next lab.

While waiting for the gel, peer edit your lab partner’s introduction paragraph, following the rubric.

Observation/Data/Results:

Order of wells: Practice, 5 uL of adder, 10 uL of sample, 10 uL of positive control, 10 uL of negative control, practice, 10 uL of negative control, practice

Our gel right when we put it in the box

Our gel after a few minutes in the box

Gel after 30 minutes in the box

Interpretation/Conclusion/ Next Steps: In the next lab , we will be able to observe the DNA in the gel. Additionally, we will compare our sample to the ladder. We will look at the gel under ultraviolet light.

Sample Storage: We placed out gel into the cardboard box on top of the 6.

Date of Experiment: 22 February 2018

Experiment Title: PCR and Agarose Gel

Rationale/ Purpose: The goal of this lab is to create a negative and positive control tube along with our soil DNA tube. In this, we will add primers for amplification. Additionally, we will prepare an agarose gel with wells to place the DNA solution in.

Procedure: Add 12.5 uL of 2X Master mix, 1 uL of cox1 primer, and 11.5 uL of deionized water into a tube. Label this tube the negative control and put group number on it. Add 12.5 uL of 2X Master mix, 5 uL of the positive DNA template, 1 uL of cox primer, and 6.5 uL of deionized water into another tube. Label this tube the positive control and put group number on it. Add 12.5 uL of 2X Master mix, 5 uL of the environmental DNA template, 1 uL of cox primer, and 6.5 uL of deionized water into another tube. Label this tube the soil sample and put group number on it. Place the three tubes into the rack.

Add 0.6 g of agarose into an Erlenmeyer flask. Pour 40 mL of 1xTAE gel buffer. Cover the opening with weighing paper and place the cap on top. Microwave the solution for 1 minute 20 seconds and then place it into a water bath to cool. While the solution is cooling, prepare the gel electrophoresis box. Seal the open ends. Add 2 uL of ethidium bromide and pour the agarose gel into the box. Place the comb in the solution and let it solidify. After it solidifies, remove the comb and pour 1xTAE gel buffer on top until the gel is covered. Label the box and store it for next lab.

Observation/Data/Results: The concentration of the DNA was 80 ng/uL and the final amount of DNA in the reaction was 400 ng. The concentration of the primers in the stock tube was 100 uM, and in our final reaction, the concentration was 0.4 uM.

Interpretation/Conclusion/ Next Steps: In the lab, we calculated the amounts of each solution to put in the tubes and the purpose of each of the solutions. Additionally, this lab prepared us for the gel electrophoresis for next lab. In the next lab, we will place the DNA solutions we created into the wells of our gel and run electric currents through the gel to create bands in the gel. Then, we will view the bands.

Sample Storage:  Negative control: -, 6; Positive control: +, 6; Soil DNA tube: S, 6. We placed it into the rack and wrote LBP on the paper besides the rack. We also labeled our gel electrophoresis box group 6 and LPB.

Date of Experiment: 15 February 2018

Experiment Title: E.Z.N.A Tissue DNA Protocol

Rationale/ Purpose: The goal of this experiment was to isolate the DNA from the cells we obtained from the last experiment. Also in this lab, we learned the purpose of each of the solutions we added to our sample.

Procedure: In our experiment, no change was made to the protocol given except for when we had to spin the Ludox cell layer for 5 minutes at 3000g. Instead, we spun the tube at 13000g.

Spin the tube for 5 minutes at 3000g (we spun at 13000g). Remove the supernatant and add 1 mL of PBS and resuspend using a vortex. Spin the tube for another 5 minutes at 3000g and remove the supernatant. Add 100 uL of PBS into each tube and vortex. Combine the cells into one tube using a pipette. Obtain 20 uL of the cells and place it in a separate tube. Add 20 uL of Iodine and observe 5- 2uL drops under a microscope.

Add 25 uL of OB Protease Solution to the cells and vortex. Add 220 uL of the BL Buffer and incubate the tube at 70°C for 10 minutes in the heat block. After 10 minutes, add 220 uL of ethanol and vortex. Place this sample into a collection tube with a HiBind DNA Mini Column. Centrifuge the tube at 10000g for 1 minute. Throw away the liquid in the collection tube and add 500 uL of HBC Buffer into the column. Centrifuge the tube at 10000g for 30 seconds. Throw away the liquid in the collection tube and place the column into another collection tube. Add 700 uL of DNA Wash Buffer and centrifuge at 10000g for 30 seconds. Throw away the liquid in the collection tube and add another 70 uL of DNA Wash Buffer. Centrifuge at 10000g for another 30 seconds and throw away the liquid in the collection tube. Place HiBind DNA Mini Column with the empty collection tube back into the centrifuge for 2 minutes at 10000g.

Place the HiBind DNA Mini Column into a labeled 1.5 mL microcentrifuge tube. Place 100 uL of Elution Buffer from the heat box (at 70°C) into the tube and let rest for 2 minutes. Centrifuge for 1 minute at 10000g. Throw away the column and place the labeled tube in the rack.

Observation/Data/Results: When observing the cells with the iodine under the microscope, we found many cells of different sizes and shapes. Our group (group 6) was unable to do a cell count because there were too many cells (>100) in each drop.

Interpretation/Conclusion/ Next Steps: In addition to following the protocol given, we were able to gain a better understanding of the procedure we were following and why we added certain solutions. By spinning the tube at 13000g rather than 3000g could possibly create an error in our overall experiment. In the next lab, we will look at the DNA we extracted from the cells and possibly do a gel electrophoresis.

Sample Storage:  We labeled our tube with our group number (6), section (22), and initials (LBP).