Carter Pope

Professor John Paul

Molecular Ecology

10/28/18

Lab 9 Entry

PCR Reactions for Plant DNA

 

On October 24th, I arrived at the lab and continued with my focus on the plant DNA. My lab group and I began by obtaining our test tubes of our three samples of different plant genomic DNA and went to the corner to begin the gel electrophoresis. Kayla and Jinwoo used a micropipette to transfer 1 microliter of loading dye and 3 microliters of each person’s personal sample of DNA onto a piece of parafilm in order to make a total of 12 little 4 microliter droplets. These droplets were recorded on a separate piece of paper for later identification purposes and then the droplets were transferred into separate wells inside the gel. Once everyone had their droplets in the wells, the gel was then run for about 20 minutes at around 140 volts. Once these gels were completed, they were taken to the back machine to be analyzed by our professor.

Thought: I couldn’t find the picture of my gel on canvas, but I know that one of Kayla’s samples and one of Jinwoo’s samples didn’t work as well so we removed those from our batch, along with a few other samples that didn’t work from other groups at the lab. Peter and I were fortunate enough to have our samples working and fully visual on the screen.

I then shifted my focus to plant DNA PCR and prepared two additional tubes for each of my three samples. I began by acquiring these 6 tubes and correctly labeling them with the sample code and pipetted 20 microliters of the correct template DNA into each.

Next, I began setting up the EPIC PCR and acquired 8 Mimulus cardinalis DNA samples at random and also got a strip of eight 0.2 microliter tubes. I labeled each of these tubes with the sample code, the primer number that corresponded to our group (5334), the date, and the tube number on the top lid. I transferred 1 microliter of each different DNA sample into the correctly labeled 0.2 microliter tube being sure to change each filtered tip between samples. I then placed the strip of the eight tubes on ice.

Jinwoo then began making our group’s master mix, which was designed to be used for about 40 samples (even though we had a total of 32 samples to do), just in case someone accidentally took too much during their transferring. I then transferred 19 microliters of the master mix into each of the eight tubes (also slowly drawing the mixture up and down the tip with the pipette to mix the solution) being careful to change the filtered tip each time just to be safe. Once each tube contained 1 microliter of its correct DNA and 19 microliters of the master mix, the eight tubes were then closed (I didn’t have to use the centrifuge because my solutions in the tubes didn’t need to be spun down) and put into the PCR machine.

On October 9th (Monday) my group and I ran a quick gel electrophoresis for our DNA samples from the eight tubes to prepare for our professor.

Carter Pope

Professor John Paul

Molecular Ecology

10/19/18

Lab 8 Entry

Plant DNA Extraction

 

On October 17th, I arrived at the lab and began our protocol for the plant DNA extraction. I started by obtaining three random capsules with different plant tissues and silicon pellets in each of them from my professor. I then labeled three 2.0 ml tubes with my initials and the plant sample code that was given on the capsule of each of the three plant variations (JP1231, JP1298, and JP1229). Three small sterile 3.2-mm stainless steel beads were added into each of the newly labeled 2.0 ml tubes. The size of about a fingernail of each of the three plant samples was then transferred with a sterile pair of forceps into the correct corresponding 2.0 ml tubes that were labeled with the sample code, along with the steel beads. Between each plant sample transfer, I wiped the forceps with 70% ethanol to ensure no cross contamination was occurring.

My lab group and I then brought each of our three 2.0 ml tubes over to our professor in the corner of the lab, where he showed us how to assemble the modified reciprocating saw in order to crush the plant tissue with the steel beads during the vibrations. I then took my three tubes over to the centrifuge and had them spun for about 15 seconds at a fast speed. Next, I brought my tubes back to the table and added 434 microliters of preheated grind buffer into each tube using a p1000 micropipette and filtered tips and then brought these tubes to a hot water bath to be incubated for ten minutes total and inverting the tubes every three minutes to mix the contents inside.

After the ten minutes passed, I brought the tubes back to my table and added 130 microliters of 3M ph 4.7 potassium acetate into each tube with a micropipette and filtered tips. Then I inverted the tubes and placed them in the centrifuge to be spun at maximum force for 20 minutes, making sure my lab group balanced out our tubes in the centrifuge. I then brought these tubes back to our table and acquired three new 1.5 ml tubes from our professor and labeled them with our initials and plant sample codes. I then used micropipettes and filtered tips and transferred out only the supernatant (avoiding any precipitate from inside the tube) from the 2.0 ml tubes that I just took out of the centrifuge and put them into the correct corresponding 1.5 lm tubes that we just got. Since each of my supernatant levels for each plant sample code measured out to 400 microliters, I then added 600 microliters of binding buffer to each of the 1.5 ml tubes (I added 1.5 volumes of the binding buffer of my total measurement of supernatant that I acquired).

After that, I got three new Epoch spin column tubes, initialed and labeled each with the three different sample codes. I transferred 650 microliters of each of the mixtures from the 1.5 ml tubes into the three new and correct corresponding Epoch spin column tubes.  I then centrifuged these tubes for ten minutes and removed the waste liquid that settled to the bottom of the tubes into a waste beaker. I then put the remaining mixtures from the 1.5 ml tubes and put them into the same corresponding Epoch spin column tubes to centrifuge again for ten minutes and emptied out the waste liquid that settled at the bottom. I added 500 microliters of 70% ethanol to each of the Epoch spin column tubes and centrifuged them for eight minutes. After the eight minutes, the waste liquid at the bottom was removed into the waste beaker. I then once again added 500 microliters of the same ethanol to the Epoch tubes and centrifuged them for eight minutes once more to collect and remove the waste liquid. Then I removed the “flow-through” parts of the Epoch tubes and kept the “column” parts.

Once this was done, I took my three “columns” to the centrifuge and spun them for five minutes to remove any residual ethanol from the previous steps. The “columns” were then placed into three newly initialed, sample coded, and dated 1.5 ml centrifuge tubes. I added 100 microliters of preheated pure sterile water into each of these 1.5 ml tubes and let them sit for five minutes. Then I centrifuged the tubes for two minutes at high spin to elute the DNA. The tubes holding our plant DNA was then given to our professor.

Thoughts: I hope our professor lets me use the modified reciprocating saw rack for later experiments.

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.

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.

 

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.