Lab 7 Entry: Phylogenetic Inference

Carter Pope

Professor John Paul

Molecular Ecology

10/16/18

 

Lab 7 Entry

Phylogenetic Inference

 

 

On the 10th of October, I arrived at the lab and opened up Geneious on my laptop to make sure I had my COI sequences set up and aligned. After that, I proceeded to attempt downloading the “jModelTest2” and using Java; however, I ran into some technical difficulties and had to fast forward that step. This step would have allowed me to determine which model would have been best for my specific set of data and presented an accurate depiction of my data in a tree. I ran a Bayesian inference on my data by using “MrBayes” and used the generic settings with “HKY” since I had to skip the “jModelTest2.” I first tested with ‘Unconstrained Branch Length: Exponential (10), and Shape Parameter (10) to ensure my analysis was incorrect. I then fixed my parameters and got a gorgeous looking analysis. Next I used “RAxML” and tested out what my data looked like using ‘bootstrapping trees.’ After that I used “PHYML” and examined my data in this analysis. Even though I had some setbacks in the beginning, I was able to do a hard restart on my poor laptop and persevere through.

I will then run another “MrBayes” on my data but with different settings and create my best tree to show next lab on Wednesday the 17th of October.

Lab 6 Entry: Introduction to Geneious

Carter Pope

Professor John Paul

Molecular Ecology

10/9/18

 

Lab 6 Entry

An Introduction to Geneious

 

On the 3rd of October, I continued to work on the sushi project in the lab. I received my DNA barcoding sequences for my fish from Canvas and began the using the program called Geneious. Once Geneious was installed into my laptop and my DNA barcoding sequences were downloaded into Geneious, I began going through the tutorial handout on how to use the program. I learned how to do many tasks, such as creating new folders, reading forward/reverse sequences, and “BLASTING” my sequences.

From Exercises listed in the handout:

  1. Since my first fish sample (Pacific Rock Cod) didn’t work during the first round of the PCR, I used Peter Dubois’ Pacific Rock Cod sample (since we got the fish at the same grocery store) and discovered that we had a 99.4% pairwise value with Sebastes mystinus. Sebastes mystinus’ common name is the Blue rockfish. The description of the fish, which was Pacific Rock Cod, technically is an appropriate name for the fish sample because Blue rockfish falls under the category of Pacific rockfish, aka Rock cod.  The second fish sample (Dover Sole) had a 99.5% pairwise value with Microstomus pacificus voucher. Microstomus pacificus voucher’s common name is Dover Sole and matched the species that was labeled in the grocery store. The third and final fish sample (Ahi Tuna) had a 99.8% pairwise value with Thunnus albacares. Thunnus albacares’ common name is yellowfin tuna. Both yellowfin tuna and bigeye tuna are the two species that are classified under ahi tuna, so technically my fish sample was correctly labeled in general.

 

 

2.   For PRCD (Peter’s Pacific Rock Cod and Sebastes mystinus), there were four polymorphic sites in the alignment of the two located on the columns: 6, 9, 573, 615.

For CPO2 (Dover Sole and Microstomus pacificus voucher), there were three polymorphic sites in the alignment of the two located on the columns: 161, 203, 596.

For CPO3 (Ahi tuna and Thunnus albacares), there was one polymorphic site in the alignment of the two located on the column: 3.

 

Thoughts: I didn’t have more than ten polymorphic sites in my alignments which could suggest that my sequences were very short and if I had captured a longer sequence with no cuts previously made, I may have seen more polymorphic sites.

 

I then made a new alignment with included my three samples (including Peter’s PRC0) and 25 other sequences of different Actinopterygii that had the COI gene and had a similar length of around 500-700 bp, as well as an additional Chondrichthyes which acted as an outgroup for my phylogenetic tree that will be created next Wednesday on the 10th of October.

 

Lab 5 Entry: Field Trip to Alpine Dam/Mt. Tamalpais/Coast

Carter Pope

Professor John Paul

Molecular Ecology

10/2/18

 

Lab 5 Entry

Field Trip to Alpine Dam/Mt. Tamalpais/Coast

 

On September 26th I arrived at the lab and our class walked to the parking structure at Koret Gym. From there, we split up into two separate vehicles and drove to the Alpine Dam in Bolinas.

Picture Credit: Peter Dubois

 

Once we reached the dam, we proceeded to walk down a path which led us to the riverbed that the dam blocked. We searched here because the ground here contained the most moisture and the Mimulus cardinalis thrives in soil that is adjacent to water. Although we weren’t able to locate any of the Mimulus cardinalis, we did find a large population of ladybugs and got to venture a little off the beaten path. One student (Jinwoo Kim) acquired a few casualties on his right lumbrical muscles on his hand. A first aid kit was applied when we returned to the lab.

Picture Credit: Peter Dubois

 

We then walked back to the vehicles and drove further into the region of Mt. Tamalpais. We then parked in a little pullout area and our professor showed us his “go-to” location for Mimulus cardinalis. It was located in between two conjoining hills where water from the top flowed downhill to form a little spring. This was an example of how the plant grew in soil that was moist with water.

 

After taking pictures, we then drove west towards the coastline and traveled just south of Stinson Beach to a pullout area for cars. We got out and walked on a trail heading to the cliffside overlooking the ocean. We set our focus back to the purple-flowering lupines and were able to find some adjacent to the path we were walking on.

Picture Credit: Peter Dubois

 

After successfully observing both the Mimulus cardinalis and the purple flowering lupine, we drove back to the parking lot at Koret Gym and ended the field trip.

 

Thoughts: The views and scenery that we were able to see and take pictures of were very fascinating and unbelievable to be quite close to USF.

 

Lab 4 Entry: Field Trip to Pescadero State Beach

Carter Pope

Professor John Paul

Molecular Ecology

9/25/18

 

Lab 4 Entry

Field Trip to Pescadero State Beach

 

 

On September 19th, I arrived at the lab at 12:45PM. We then acquired a van and journeyed to Pescadero State Beach, south of San Francisco along the coast about 50 miles in hopes of seeing the yellow flowering Lupinus arboreus. Our professor then brought us towards the cliffside with sandy soil facing the ocean and showed us an example of Lupinus arboreus. These plants were scattered here and there along the cliffside but not in abundant numbers. There were no yellow flowers visible on this plant but it was still alive and well.

 

We then walked across the bridge and then under it in order to reach the other side of the road which was more inland. Here we also saw some Lupinus arboreus but in a little less sandy of soil. We were able to find some of the plants with flowers.

 

Other Lupinus arboreus were also located and we took pictures of them. They were about three to four feet wide and about two to four feet tall as mature plants.

 

After observing these Lupinus arboreus plants, we then headed back to the van and journeyed north towards San Francisco. We planned to also visit another beach ( I believe Pomponio State Beach) but due to time, we decided not to stop and look for more Lupinus arboreus because we risked getting caught in traffic. We arrived back at school around 4:45PM.

 

Lab 3 Entry: The Sushi Test (continued)

Carter Pope

Professor John Paul

Molecular Ecology

9/18/18

 

Lab 3 Entry

The Sushi Test (continued)

 

 

On September 12th, I returned to the lab and retrieved my PCR tubes that I put in the thermocycler on September 5th. Our group then brought our PCR tubes to the side benches in the lab and began setting up a new gel in order to do gel electrophoresis. One of my members (Kayla) used a micropipette and made four rows of three of 2 microliters of loading dye onto a small piece of parafilm. Kayla then added 3 microliters of each person’s PCR tubes in the correct corresponding rows of the droplets of loading dye, making sure to use a new pipette tip each time. I then used a microliter to transfer the 5 microliter samples from the parafilm into the wells of the gel, making sure to use different pipette tips each time and that the wells were labeled on a piece of separate paper and each person’s samples could be identified. Gel electrophoresis was then conducted for about 15 minutes.

Once approximately 15 minutes passed, our group collected the gel and took it to be observed under the machine for special imaging. The image showed that one of my group members had all three of their samples, two of my three samples worked (CP02 and CP03), and two of my members, unfortunately, had none of their samples work. My wells were the three on the far right on the top row.

 

Since two of my three samples worked (CP02 and CP03), I then continued with the ExoSap PCR Clean-Up protocol. I collaborated with a group of fellow classmates whose samples also worked. A master mix of containing water, 10x buffer, SAP, and Exo was made for 20 reactions and its combined total measured 250 microliters (12.5 microliters per reaction) and placed in ice. A long series of new PCR product tubes were provided by the professor and each of us received a certain number of these tubes (since I had two samples that worked, I got two tubes; my tubes were #10 for CP02 and #11 for CP03). I labeled and recorded my tubes with the appropriate identifications and proceded to transfer 7.5 microliters of my CP02 into #10 and 7.5 microliters of my CP03 into #11. Then I transferred 12.5 microliters of the ExoSap master mix into #10 and another 12.5 microliters into #11 (all while using clean pipette tips in between each transfer of liquids). Once each person finished establishing their PCR tubes, we all put our tubes together in a thermocycler. The lab period then ended and we were excused to leave. Our professor then put the tubes into a labeled tube rack and placed into the freezer.

On September 26th, we will continue with this lab and start the EXOSAP program.

 

 

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