Lab 12 Entry: ISSR Analysis

Carter Pope

Professor John Paul

Molecular Ecology



Lab 12 Entry

ISSR Analysis


On November 14th I arrived at the lab and began by adding 1 microliter of loading dye to each of my 15 PCR tubes with a micropipette. I then went over to a large 2 % gel and a lab mate and I (Jinwoo) transferred 5 microliters of each of our samples into individual wells, making sure to change out pipette tips each time and that we recorded where our specific samples were located on the large gel. Once everyone in the lab transferred their samples into the large gel, the gel was run for a long time on a low voltage.

I then turned on my laptop and loaded Geneious up onto my screen. Unfortunately, our lab table’s DNA showed no success, so our professor had us use a different set of DNA and assigned us with a new marker (5551) to use as a replacement. Since we got new DNA and marker, we redid steps from a previous lab dealing with coding heterozygotes. I made a new folder in Geneious, downloaded, and extracted the DNA set for ‘5551’ from canvas to my folder and made an alignment using ‘Muscle’. I edited any bad parts of the alignment and then located any heterozygotes that needed new ambiguity codes. The next thing I did was select each forward and reverse pair for a single sample and make a consensus sequence for each one. After making sure each of these had correct editings and base calls, I created a sequence list with all of the consensus sequences that I made with each pair. Then I added in the two samples that didn’t have a pair (JP1314 and TG0248) with the sequence list and made an alignment with all of this using ‘Muscle’. Making sure edits were made on any necessary trimmings of the alignment, I then named it “5551_Nucleotide alignment_Carter” and put this into a new folder labeled as “Concatenate.” (I must’ve read past the next step during lab, so a few days later I exported this alignment from Geneious into canvas under the assignment called “EPIC ALIGNMENTS”)

I then went to canvas and chose three other marker alignments (making neither of them were from the same marker) and downloaded/exported them into my Geneious folder called “Concatenate.” I then selected these three marker alignments, along with mine, and made a concatenate alignment. Further use of this concatenate alignment will be used to create analyses and phylogeny trees in further assignments.

Lab 11 Entry: Lupinus arboreus DNA PCR

Carter Pope

Professor John Paul

Molecular Ecology



Lab 11 Entry

Lupinus arboreus DNA PCR


On November 7th, I arrived at the lab to shift my focus towards running a PCR with ISSR primers for some Lupinus arboreus DNA samples that were from a previous class. I started by making three dilutions for each of my five Lupinus arboreus that I was given (making a total of 15 dilutions). In order to do this, I acquired fifteen 1.5 millimeter tubes and used a sharpie to label each tube with its correct sample ID and ‘1:10’ (each of the five samples had its unique ID). I then used a micropipette to transfer 45 microliters of water into each of the 15 labeled tubes. Next, I used a micropipette to transfer 5 microliters of the correct DNA sample into each of its three corresponding 1.5 millimeter tubes (making sure to change tips each time). Once my group completed these steps, we then put our dilutions into the correct tray for our professor to disperse evenly for each of the three different lab groups.

After our professor gave the lab groups the trays of every diluted sample, my lab table peers and I chose 3 different sets of the five DNA samples at random to work with a total of fifteen Lupinus arboreus DNA samples (For example, I chose CRA01-CRA05, PRK01-PRK05, and PSF01-PSF05) and recorded their unique ID and the date down on a piece of paper. I then got two strips of eight 0.2 millimeter tubes and labeled each tube lid with a number (starting with ‘1’ for the first tube and increasing in value with each tube until reaching the last tube and labeling it ’16’). I then labeled the side of each tube with the unique ID and put both my initials (CP) and our primer name (17898) on the side of the strip of tubes. I used a micropipette and filtered tips (making sure to change them after each transfer) to then transfer 1 microliter of each of 15 different diluted DNA samples into each of the correct corresponding 0.2 millimeter tubes that I just labeled in the previous step. Once I finished transferring the 1 microliter into the tubes, I put my tube strip on ice until my lab group finished.

During this time, a person in my lab group (Kayla V.) made a master mix for 80 reactions (60 for our group’s total number of samples plus an additional 20 just in case). I then used a micropipette to transfer 19 microliters of the master mix into each 0.2 millimeter tube in the strips (being careful to change the tips each time) and carefully mixed the solution by pipetting up and down slowly in the tube. The tube stips were then given to our professor, who would then put them into the PCR machine.

Lab 10 Entry: Analysis of EPIC markers

Carter Pope

Professor John Paul

Molecular Ecology


Lab 10 Entry

Analysis of EPIC markers


On October 31st, I arrived at the lab and was given the sad news that none of our class’s plant DNA worked. Instead, we used a previous class’s plant DNA as a temporary replacement to continue our work with Geneious. I began by opening Geneious on my computer and making a new folder to hold my EPIC sequence reads, both the forward and reverse sequences. After importing these sequences from Canvas to my new folder, I then built an alignment using Muscle and began trimming the bad reads at the beginning and end of the sequences.

Once these edits were made, I zoomed in and began looking for polymorphic sites, heterozygous sites, as well as sites that were both heterozygotes and had polymorphisms. Polymorphic sites could be found in my following columns: 50, 91, 106, 131, and 411. Heterozygous sites could be found in my following columns: 271, 391, 403, 412, 457, 458, 465, and 468. Sites that were heterozygous and polymorphic were on columns 403 and 412. The heterozygous sites were then edited to match the correct IUPAC Ambiguity Code.

The next step I did was to create consensus sequences from the forward and reverse reads. I began by selecting the forward and reverse pair for each specific DNA code and aligned them to make a new assembly document. I edited out bad sections of the new aligned sequences, changed any obvious unknown bases that were labeled “N”, and then saved the changes. I did these previous two steps for every individual DNA code that had a forward and reverse read (TG0248 and JP1132 were the only two that didn’t have both the forward and reverse read). Then I selected all of my newly edited assemblies and created a new consensus sequence list. I extracted this sequence list to a new subfolder and labeled it “5334 Consensus Sequences”. I then edited this “5334 consensus sequence” with a “Batch Rename” and put 34 in the text space provided in order to remove all the characters except the sequence name for the selected documents which is needed to concatenate the alignments of different markers. However, after working with Professor Paul and Jinwoo Kim during office hours, we discovered that selecting 34 would result in the four number on the far right on the identification code being cut off. This lead to a problem because several DNA samples now had the same code and they now couldn’t be distinguished. Therefore, I redid the steps starting from extracting the sequence into a new subfolder and this time labeled it “Correct 5334 Consensus Sequences” and put 33 in the text space provided. This fixed our problem and I continued by adding the TG0248 and JP1132 which were the two that didn’t have both a forward and reverse read, and added them into the “Correct 5334 Consensus Sequences”.

I then built an alignment with Muscle using the default parameters for the “Correct 5334 Consensus Sequences” and made a last minute edit towards the end of the alignment which cut off a few hundred base pairs that looked bad. I saved these edits and then renamed it as “5334 Nucleotide Alignment”.

At home, I continued working in Geneious by inferring a Bayesian tree of this single locus alignment. By using my “5334 Nucleotide Alignment” and choosing MrBayes to run a tree, selecting M. lewisii (TG0248) as the outgroup, and running the tree long enough, I was able to get a good posterior distribution and fuzzy caterpillar.

The tree on the left shows equal branch length and the one on the right shows proportional branch length.

My laptop is very difficult in cooperating with me when it comes to transferring over images and dealing with big images that hold a lot of memory so I had to take a picture of my laptop screen with my iPhone. Link: 5334 nucleotide tree-288bbml

Based off of the trees that I created, I can infer that there are many clades that are strongly supported through the MrBayes that I ran. The clades that are formed support the idea that they indeed represent geographic populations for the most part. The clades will show that the plant species in each clade are in a similar geographic setting; however, other clades can possibly also be in a generally similar geographic setting, regardless of them being in a different clade.


Below shows my posterior distribution and my trace:


Thoughts: For some reason, I can’t seem to find my options for working with my nodes and even changing the decimal place for them won’t change when I try editing the settings. I might have to go in during office hours and find any potential mistakes that may have occurred.