Phylogenetic Analyses of Species in Thomasomys (Rodentia: Cricetidae)

Picture for Blog Entry

My research experience with the biology department has certainly been a memorable part of my time here at ACU. Starting in January, I jumped in on a project of Dr. Lee’s, a portion of which he delegated to Dr. Brokaw – the continuous updating of an exhaustive taxonomic tree of a rodent genus living in Ecuador named Thomasomys. This genus of mammals living in Andes Mountains has undergone rapid adaptive radiation, occupying a variety of niches, differing in climate and elevation. Dr. Lee and Dr. Brokaw are attempting to piece together the evolutionary relationships among species within this genus.

Here are pictures of our phylogenetic trees from two different genes, cytochrome b and CO1 (both mitochondrial).

Published Image

This image includes DNA we sequenced in the spring of 2015 and was featured in a publication by Dr. Lee and Rachel Ritchie. Before getting included in publications, any DNA we sequence is submitted to GenBank and becomes accessible to researchers worldwide.

CO1 ML Tree With Bootstrap Values 27-Nov-15

This image is a phylogeny based on our latest sequencing of the CO1 gene.

These are our results after the meticulous processes of running PCRs, gel electrophoresis, DNA purification, DNA quantification by NanoDrop, and sequence editing and alignment. During my participation in this project, we added two new species to our trees, T. ucucha and T. rhoadsi, found out that T. cinnameus may actually consist of two different species, and elucidated the confusing geographic distributions of the closely related sister species, T. baeops and T. taczanowskii.

Although the principal goal of the project isn’t entirely fascinating to me nor directly relevant to my future career, the experience I’ve gained working in the lab is something that I can take with me for the rest of my life. Extracting DNA, using a thermal cycler during the PCR process, running an electrophoresis gel, purifying DNA, and editing sequences have all been extremely useful techniques that I’ve learned. It’s also been fun getting to work with fellow classmates, and I learned a lot from other students like Tina Johnson, who was Dr. Brokaw’s lab mentor in the spring. Also, presenting at the ACU Research Festival was a fantastic experience, even though it was a little nerve-wracking.

One of the difficulties that I initially came across was the fact that I had no idea what was going on scientifically when I first started. I didn’t even know what “PCR” stood for until the day before presenting at the festival [The polymerase chain reaction is used to replicate DNA]. Creating a “master mix” for the PCR and loading the gel are meticulous processes that require complete focus, and I made silly errors at first because I wasn’t careful enough. This semester, my group had to repeat two steps, PCR and gel electrophoresis, multiple times, and it felt a little rote at times. We made a lot more progress in the spring semester, probably because the goal of presenting at the ACU Research Festival was more imminent. I also learned that research doesn’t turn out the way you plan it a lot of times – things go wrong more often than expected, like the way our PCR’s have turned out this semester.

Doing research while taking classes such as Cell Biology, Biostatistics, and General Biology II is a great way to apply what you’ve learned in a hands-on way. It’s also a great way to get to know the professor that you’re working for and to have the opportunity to ask him or her questions outside of the classroom. I believe that doing research for the biology department at ACU is an experience you don’t want to miss.

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Reflections on 2013/2014

It’s hard to believe the school year has finally come to an end—and even harder to believe that I began working on my summer research project about a year ago (oh, how the time flies). At the beginning of the summer, we were designing primers, and now we’re in the process of identifying specimens using SNP haplotyping. While we haven’t made as much progress as we initially envisioned (due to technical difficulties, among other things), we still identified quite a few and have made quite a dent in this continuing project (which we look forward to working on more). If nothing else, analysis of our soil collections suggested edaphic specialization in Mentzelia monoensis, which was a really exciting result to share with the world.

This semester I was not able to spend as much time in the lab, but I did present a 15-minute oral presentation at 3 different conferences. In January, we (Dr. Brokaw, Christian Hofsommer, and I) presented our work in Austin at the conference for the Society for Integrative and Comparative Biology. It was scary presenting my work in front of an audience—but it helped me to realize that I am fully capable of presenting information to a scientific community. In March, I  (in addition to several other biology and English students) presented at Alpha Chi’s National Conference in St. Louis—which was quite different from the Evolution and the SICB conferences. This conference focused primarily on undergraduate research and had presentations in over 26 different categories! So, it was really a unique experience to be exposed to so many different disciplines. I shared my work to an audience (that included my wonderful friends!) during the Organismal and Ecological Biology section. Elisa Wyrick (who researches British Literature) and I won best presentations in our categories—which came as quite a surprise! Finally, I presented my work for the last time at the ACU Undergraduate Research Festival at the beginning of April. By the end, it felt like I had come a full circle–presenting my research to my teachers and peers. Additionally, we (Dr. Brokaw, Christian Hofsommer, and I) are working on a publication, so stay tuned for more on that!
Ultimately, I feel I learned quite a lot during this past year. Slowly (but surely), I’m morphing into a scientist: I’ve learned to think more critically, ask more questions, and take more risks. And, as I’ve mentioned before, I’ve learning that failure is part of the process. Things don’t always turn out the way we’d like them to—not to mention everyone messes up from time to time. I definitely didn’t envision the primers or the specimen from different herbaria to give us difficulties, but unfortunately they did, and we had to troubleshoot around them. Despite these challenges, I’ve developed more independence and confidence in my research abilities as well as myself. I never could have imagined I would be able to explain my ideas to a room full of people—yet here I am on the other side.

A year later and I have presented at 3 national conferences, conducted field work, experimented with new methodology (primer design, allele-specific PCR, etc.), and won an award for my presentation (this one I still can’t believe)—not to mention I made many new friends! It’s difficult to articulate really just how much I have benefitted from this entire process, and I am so grateful for the opportunities research has given me (and many others) during my time at ACU. Unfortunately, this summer I will not be continuing my M. monoensis work, but instead I will be researching molecular and cell biology at Pepperdine! It’s a bit intimidating to be moving into a new lab with new people, ideas, and methodology—but ultimately, I know my experiences at ACU have well prepared me for this new adventure. Also, during the fall I will be working as a research tutor—which I honestly couldn’t be more excited about. It will be truly awesome to teach other students about research and share my passion with them.

All of the ACU Alpha Chi members attending the conference in St. Louis!
All of the ACU Alpha Chi leadership attending the conference in St. Louis!
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Bat Research Class

I began research with Dr. Brokaw and Dr. Lee at the beginning of Fall 2013. They wanted to start an entirely new project with me, so a lot of the beginning stages were simply figuring out what to research. Our options were really between rats and bats that Dr. Lee had collected. So, we went down to the natural history collection and looked at what species Dr. Lee had collected a lot of. He had a lot of Mexican free-tailed bats, or Tadarida brasiliensis, that he had been wanting to do a project with, so we ended up designing an experiment around that. Since he had samples from both Ecuador and Texas, two geographically distinct areas, we decided to see if there was any genetic variation between the two populations. Dr. Brokaw taught me DNA extraction and PCR first. However, we had quite a bit of trouble getting a good PCR for the majority of the experiment. After months of not being able to figure out what I was doing wrong, Dr. Brokaw was finally able to confirm that some of the chemicals I had been using were degraded. Once we fixed that problem, my PCRs worked fine. We cleaned and nanodropped (measured the concentration) the DNA from the cytochrome b gene before sending it off to another lab to be sequenced. Somewhere along the line, I also acquired a couple of research partners on the project. However, we usually worked on different schedules and got back together to share results. At the beginning of the spring semester, I finally met my partner that I continued to work with, Lindsey Bloomer (our other partner is currently on study abroad). Another road block that we had involved one of the coolers in the lab breaking down several years ago. It turned out that many of our DNA samples from Texas got ruined when they had thawed, and we only had one sequenced sample from Texas. We had to push our project to the very last minute to fix this. I literally finished the last set of DNA extraction, cleaning, and nanodropping all in two weeks. Luckily, we got four more sequences from Texas thanks to samples from the natural history collection at Angelo State University. Towards the end of the project, we edited the sequences and constructed a haplotype network of the cytochrome b sequences from different samples, which gave us some very interesting results. The bats from North America were put into a completely different network than the bats from South America (at this point, I had pulled some additional sequences from GenBank to compare to our new samples). Overall, we ended up deciding that the Texan bats probably did not migrate to South America and vice versa because there were no shared or even similar haplotypes in both locations. We plan to continue this research next year with the focus on a different gene that mutates a bit slower to find similarities between to two groups. My favorite parts of the project were trying to extract DNA from a bat foot and being asked by Dr. Lee to look for a sick bat that someone had reported was lying on the ground by the library. I really enjoyed learning from my wonderful mentors, and I am really excited for next year.

Bloomer and Burt

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Bioremediation Research Course

Over the course of this past year I have been working with Dr. Brokaw on the effects of remaining hydrocarbons on vegetation following bioremediation of a crude oil spill. This project was renewed back in 2010 after nearly a decade of allowing revegetation to the testing sites. I was privileged enough to be allowed to come into this research at the beginning of the Fall 2013 semester to continue what the previous researchers had been working on. Dr. Brokaw and I began by testing control groups of a C25 hydrocarbon and a C36 hydrocarbon on the GCMS (Gas Chromatograph Mass Spectrometer) to get a better understanding of the unknown hydrocarbons that would be tested from the soil. We did this by taking known solutions of either C25 or C36 mixed with cyclohexane, and then we used the GCMS to measure them. After coming to the conclusion that the concentrations of C25 and C36 can be reliably measured and that they produced very similar results, we have moved on to begin re-measuring the soil samples that were collected a few years back that might have similar hydrocarbons in them. We ran into a problem just recently with the GCMS breaking down on us, but that will not be a problem for us to continue later in the summer. The amounts of hydrocarbons in the soil are measured by washing the soil with cyclohexane by use of Soxhlet extraction. I plan to begin this part of the research this summer in June after Dr. Brokaw and I travel back to the spill site to collect more samples. I am very excited and interested in the continuation and results that this research will bring.


Being involved in this research has been such a blessing by the Biology Department, but mainly Dr. Brokaw. Having a mentor as patient and understanding as him has definitely made this experience all the more amazing. I think that the opportunity to participate in graduate research and build relationships with the professors here at ACU is unparalleled and is really the ACU difference.

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Aeromonas and Research Tutors

In the last post I was about tell everyone how I got a third degree burn from making enchiladas and (this is embarrassing because I do microbiology research I should know better) how it got infected and I was out of the lab for three days. Bacteria are no joke. Who knew a tiny burn could make you so sick. It felt like I had the flu. All I did was sleep and I could not use my entire arm because the pain was so bad. Don’t do that kids. At least now I have cool pictures of all the layers of my skin growing back and scarring so that Dr. Huddleston can show her Anatomy classes. Fast forward the summer and the fall semester (I really should have kept up with this blog, whoops) and here we are at my last semester at ACU.

This semester we are welcoming two more new people to the Huddleston research group. Welcome, Sally and Kristen! Now there is total of six people in our lab. This year of research may not be as exciting as last summer when we welcomed two new people to our lab group, the Huddlestons welcomed baby Luke, and I had my near death experience, but we plan on making big progress this semester. We are a lot more focused this semester thanks to the new research class. Five members of the Huddleston lab are now also taking a research class for upper-level biology credit. This allows us to learn new techniques and have lots of a time to work in the lab. We will be attending and presenting at the Undergraduate Research Festival at ACU and at the Texas ASM conference in New Braunfels later in April.

Right now we are working on a way to get our products AB and C to form ABC using the SOE PCR technique. We have made new reagents, ran the DNA out on a gel to make sure it had not degraded, tweaked the PCR protocol, and checked the primers. Next, we plan to increase the concentration of template added to the PCR mix to see if that has any positive outcomes. Once we get the ABC product made we can incorporate it into our Aeromonas cells and get moving on research. You have to be a really patient and meticulous person to get through research sometimes. The roadblocks are discouraging sometimes, but I really want to make a lot a progress this semester. This is all the time I have left!

I plan on working on my complementation vector this week- Sing Song willing- if it doesn’t snow again like it did last week when I was supposed to work on it. The gene of interest has already been ligated into the vector. I just need to transform the vector into E.coli cells to screen them for the recombinant plasmids.

ACU Biology Research Tutors Kathryn Davis and Madeline Peterson
ACU Biology Research Tutors Kathryn Davis and Madeline Peterson

Another fun thing about this semester is that Dr. Brokaw asked Madeline and I if we wanted to be the first research tutors for the biology department. Every Wednesday and Friday one of us is in the lab to help with lab upkeep and teaching new student researchers the basics of biology research. We teach them how to run PCRs, DNA extractions, and how to run EtBr or SYBR gels. The necessity of lab tutors shows just how much the biology research group has grown in the past couple of years. Yay, biology!

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Mentzelia and DNA

During the last year, I’ve been doing research with Dr. Brokaw on the genetics of species in the genus Mentzelia. This research involves sequencing the DNA of these plants and looking for similarities and differences between multiple species in this genus in order to see if the regions we’re using are a definitive indicator of a difference between the species. In addition, we’re using it to compare a few species in particular, with a special focus on Mentzelia monoensis. This species, thus far, has only been found in one region in Mono county, California. Because the traditional means of differentiating it from other species, it’s seeds, is not always feasible depending on the season and the presence of seeds in a particular sample, it’s important to be able to identify it through alternative means. To this end, we are comparing the chloroplast DNA in Mentzelia monoensis to species that are difficult to differentiate by sight alone.

My time in the lab has primarily been dedicated to the extraction of the DNA and interpreting the results of this DNA. The first step in the process is the collecting of a sample from a pressed plant, and grinding the plant matter so that the plant cell proteins don’t interfere with the DNA extraction process. This is probably the most tedious step in the process, especially when working with many samples at once. Often the pressed samples are difficult to get any leaves from, and moving the leaves takes some precision. The grinding itself can be pretty tiring on the fingers, as the leaf samples are rather small and therefore necessitate using a very small pestle to grind them, which can strain one’s fingers after a while. Even though this step can be boring, it’s also very interesting. The samples we use often are from very different geographic regions, given to us by herbariums across the area that we’re studying. In addition, the samples are very frequently older than myself; of the oldest samples we discovered was over 100 years old!

The rest of the process often goes much smoother, with enough to do to remain interesting. After extracting the DNA by grinding it, we have to purify the DNA and then amplify it through a polymerase chain reaction that essentially copies the DNA several times. We then send the DNA off to a lab for them to sequence, and we receive the results, correct any errors, and compare them to previous samples. The entire process has been fairly exciting. It’s been great to see our results accumulating over the last six months and supporting our hypotheses about the plants we’re studying. I’ve learned quite a bit about the research process, especially how to avoid mistakes (it can be rather difficult to figure out what a sample is when you rub half the label off!), and I’ve gotten a lot of valuable lab experience working with machines and processes that I might encounter later in my life. While we haven’t been able to get many results recently because we haven’t quite mastered our new extraction process, I’m looking forward to continuing to proceed and eventually get more done. Our results have thus far been interesting, and I’m excited to see where they go in the future.

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Biology prof discovers new animal species

As originally posted in “ACU News” on October 08, 2013:

While leading a student expedition in Ecuador’s Sangay National Park in 2010 Dr. Tom Lee, professor of biology, discovered a new species of opossum. Lee, along with one ACU student and one Ecuadorian student, were the first people to ever see the species.

The shrew opossum is making its scientific debut next month. It will be listed in the Oct. 2013 issue of the Journal of Mammalogy.

“It’s exciting to go to Ecuador to see new things that people have never seen before,” says Lee. “Ecuador is one of the most diverse places in the world, and taking students there is very rewarding.”

Lee’s Ecuador discoveries can often be found in ACU classrooms. In Lee’s ecology, animal biology and mammalogy science classes, students see pictures from his expeditions, if not the actual specimen itself.

Four specimens of the rat possum belong to ACU and are used for undergraduate research.

On another Ecuador trip, Lee had photographed an unknown mammal. One year later, the animal is now recently classified as the olinguito.

Every other summer, Lee takes students to examine Ecuador’s ecosystems. The Ecuador project started in 2000 when Lee went to Ecuador with a friend who was carrying out separate research in the Galapagos Islands. The excitement of undiscovered species in the area pulled Lee to keep coming back, inviting students to learn alongside him.

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A genetically expressible summer!

This summer Dr. Hunter gave me the opportunity to join her Marine Biology research team. While Dr. Hunter is a part of a larger team in the process of assembling the tree of life for echinoderms as a whole, her research is primarily focused on determining the phylogenetic tree for Ophiuroidea (brittle stars).

I had no prior experience with research, so I had no idea what I was getting myself into when I walked into the Biology A-Lab on June 1st.  That first day, Dr. Hunter discussed what the goals for the summer were. She primarily wanted to finish getting the DNA sequences for all the specimens we had. Specifically, there were three genes that we were trying to isolate from each brittle star specimen. She had almost all of the ribosomal sequences, but she was still missing many of the mitochondrial COI gene sequences.  Initially, this task seemed very simple; however, I was very mistaken.

For the first 5 or 6 days, Dr. Hunter walked me through the different lab procedures such as PCR (polymerase chain reaction) and gel electrophoresis. She planned to teach me how to do DNA purification and DNA extraction as well, but Ethan (Dr. Hunter’s son) made his appearance a few days early. After Dr. Hunter went into labor, I attempted to isolate the mitochondrial gene in several species on my own. Amidst many failed attempts, I was able to isolate the COI gene for one species. While Dr. Hunter was gone, I did learn a lot of procedures from other researchers like Dr. Brokaw (he researches plants) and his student researcher, Tina Johnson. However, I was very excited when Dr. Hunter came back.

As the summer continued I felt like I was not making much progress with the COI gene. I tried a variety of modifications in an attempt to isolate COI, but nothing was working. About three weeks before the end of the summer, Dr. Hunter decided to set the COI gene aside and instead focus on the ribosomal genes, 16S and 18S. After this decision, my new mission was to help Jessica Bryan and Bailey Gaspard, two other members of the team, finish sequencing the 16S and 18S DNA genes for all the species we had left.  During the last two weeks I spent over 90 hours in the lab doing combinations of PCRs, gels, and purifications. By the end of those two weeks, Jessica, Bailey, and I had just about isolated and purified the DNA for all the species we had obtained. All that remained was the sequencing.


Last summer was definitely an amazing experience. I loved going to lab each day and challenging myself with each new adventure the lab held for me. I am also incredibly thankful for the friendships I made this summer! Though I learned a lot through researching, the most significant lesson I learned is that sometimes you have to sort through a lot of failed attempts to find what you are looking for.

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Students spend the summer researching

As originally posted in “The Optimist” on September 5, 2013:

By Maggie Marshall
Posted on September 5, 2013

Over 50 students from at least 12 departments conducted research across the country this summer.

Research was supported in part by grants and conducted from New York to California, covering subjects such as wildlife on the Dyess Air Force Base, the effect of having too much choice and the role of technology in higher education.

Undergraduate research projects are overseen by a faculty member and are thus often related to the interests of that professor.


“I worked with Dr. Joshua Brokaw,” said Tanner Hamilton, junior biology major from Fort Worth. “Prior to the project, I had him as a professor freshman year but didn’t know him on a personal level. Now I do and can talk to him comfortably one-on-one, first name basis.”

Dr. Jennifer Shewmaker, associate professor of psychology worked with Caitlyn Spain, junior marketing major from Denton, over the summer and is continuing research into this semester.

“We’re looking at children’s responses to gendered product packaging,” Shewmaker said. “We’re studying something called stereotype threat… the idea that we all have parts of our identity that we’re aware that other people are going to make judgments about us because of.”

This kind of hands-on research is a special experience, said Shewmaker.

“From the very beginning, she’s a part of what I’m doing and that is so rare, to get to do that.”

Much of the undergraduate research is done during the summer, but it often continues into the school year.

“We’re still working on ours,” Shewmaker said.

Students often benefit from having a faculty mentor. The wisdom and knowledge that is shared between professor and student is one rarely seen during the regular school year.

“He has been very helpful and willing to teach and instruct me,” Hamilton said.

Research reinforces classroom knowledge and ultimately leads to a better understanding of the topic.

“This is super hands-on for her,” Shewmaker said, “so she’s gaining so many valuable experiences.”

These hands-on experiences differ quite a bit from a typical lecture-based class.

“My project is a biology major specific one, specifically genetic variance in a specific genera of South American rodent,” said Hamilton. “There is no classroom involved. It’s strictly a laboratory setting with a few hours a week of active work in the lab performing experiments and interpreting data… it really helps in the application of concepts and classroom knowledge.”

From beginning to end, students are involved in these research projects. They help choose what to research, gather data, conduct tests and see every aspect of the project through to the end.

“I think it’s a huge opportunity because, for example, Caitlyn, she’s involved in every aspect, every phase of my research,” said Shewmaker.

Hamilton said undergraduate research made him commit to learning about the subject he was researching.

“It is also preparing me for next level after college because you have to hold yourself accountable. I would suggest it to anyone with any interest just to see what it’s like and to help with the comprehension of material.”

Experiences like that of Hamilton and Spain are not often found at other universities.

“It sets our graduates apart because a lot of students who go to larger universities aren’t going to get hands-on, one-on-one mentoring with a faculty member,” Shewmaker said. “It’s something at ACU that’s really special.”

Such extensive research cannot remain a secret, however. Every year, students who participated in research get to show off their hard work at the Undergraduate Research Festival.

The festival is held to celebrate and recognize the students who participated in research that year. Applicants must be current undergraduates or recent graduates from a local college. Presentations can be in the form of a paper or a poster presentation and students must fill out an application and meet certain standards to be accepted.

“I’ve been a part of the Undergraduate Research Festival since we started it,” said Shewmaker, “and it has just grown and it’s really, really fun.”

The next Undergraduate Research Festival is scheduled for April 1, 2014 and will feature students who conducted research April 2013 – April 2014.

Students interested in ACU Undergraduate Research can visit and click on Get Involved!

The festival brings people together from all disciples across the university from the sciences to literature.

“That’s what being a university is all about,” Shewmaker said. “It’s about learning and growing together.”

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SNP Haplotyping

For the majority of the summer (save for my two week adventure), I have been continuing my research with M. monoensis in the lab. Much of our prior research focused on identifying a DNA barcode, a region that can be utilized to identify an unknown specimen. So recently, we established the ndhF-rpl32 region as a DNA barcode to differentiate between M. monoensis and its relatives: a SNP (single-nucleotide polymorphism) exists within the region. Mentzelia monoensis has the “G” allele and others (such as M. montana and M. albicaulis) have the “T” allele.

With the barcode, then, identification becomes less difficult–you just need to examine the specimen’s sequenced DNA. Sequencing, however, is expensive. Often, we pay upwards of $6 for each sample’s sequence. Individually, this figure seems insignificant (I mean we pay $50 for our gel combs, right?). But given 100 or so samples, that $6 easily becomes $600. This can hamper small labs with limited funds and, in turn, our efforts to establish the true distribution of M. monoensis. Thus, using our understanding of the SNP, we decided to investigate more cost-effective methods of identification. We found one method, known as SNP haplotyping, particularly promising. This approach primarily utilizes allele-specific PCR and gel electrophoresis, which are both cost-effective procedures.

In order to begin our work with SNP haplotyping, we first needed to design primers–they would be the key to the project’s success. The process itself requires three primers total: a common reverse primer and two forward allele-specific primers. The common primer didn’t require much alteration; we just made sure it worked well with our sequence. However, the allele-specific primers required a few additions. Specifically, we added destabilizing mismatches within five bases of the 3’ ends, which facilitated primer specificity and, thus, identification.  Additionally, we had to attach 5’ tails to our primers. The lengths of said tails differed by about 10 base pairs, which allowed us to distinguish the different species on a gel. This was definitely one of the more difficult aspects of the project, especially as Dr. Brokaw and I both had limited experience with primer design. We suddenly had to consider GC content (for the Tm), primer dimers (when the primers bind to each other), and hairpin loops (when the DNA folds back on itself) among other things. Though with the help of Dr. Huddleston (she’s awesome!), we were able to design a common primer and 7 (yes, 7) potential primer pairs. Why 7? Well, often (as I now understand) primers tend to be a mixed bag. Sometimes they work and sometimes…not so much.

Once we received our primers, we began testing them using DNA from a few samples of M. montana, M. monoensis, and M. albicaulis. We ran many PCRs and many gels. With those PCRs and gels came many failures and disappointments. We soon discovered that most, if not all, of our primers behaved inconsistently. Some primer pairs didn’t even amplify at all! And don’t even get me started about our negative controls. In fact, until about two weeks ago, we weren’t quite sure if this project would have a “successful” result. But, lo and behold, we found hope in primer pair 3 after testing a few of the primer pairs again. The M. monoensis samples (whose AS primers had shorter tails) traveled noticeably further than the M. albicaulis or M. montana samples. So, what was so special about primer pair 3? Well…we’re not quite sure. But primer pair 3, interestingly enough, was the only pair to have 2 strong mismatches (ie a purine replacing a pyrimidine, etc.). All the other pairs had combinations of weak (ie a pyrimidine replacing a pyrimidine, etc.) and strong mismatches. In the future, it would be interesting to design primer pairs with only strong mismatches–perhaps more of our primers would work? But quite honestly, I am thrilled that primer pair 3 even cooperated at all.

Primer Pair 3
Order (from left to right): 1. M. monoensis 547 , M. monoensis control, 2. M. monoensis 554, M. montana control, 3. M. monoensis 556, M. monoensis control, 4. M. monoensis 557, M. montana control, 5. M. monoensis 558, M. monoensis control, 6. M. monoensis 559, M. montana control, 7. M. monoensis 560 (didn’t amplify), M. monoensis control, negative control (with faint band)


Along the way, we made some important discoveries about some of our materials and methods. Early on we made the switch from using TAE buffer to TBE buffer, as some suggested TBE worked well with smaller products (ours are around 200 base pairs) and produces better band resolution. Next, we transitioned to MetaPhor agarose because it can distinguish small PCR products and improve the clarity of the gel. Despite the extra expense, it seems to be working better than the standard agarose (for our purposes at least). Our most important change, by far, was our transition from SYBR-Green to ethidium bromide (SYBR-Green is people!).  Not only that, but now we stain our gels after they’ve run (another change: gels now run in the refrigerator). It is a bit scary handling a carcinogen, but ethidium bromide has facilitated the project. Also, we have been tinkering with the voltage and length of our gels. Initially, we tried smaller (about 40 V) voltages, hoping this would allow us to best distinguish between small distances. However, we now realize that “if the voltage is too low, then the mobility is reduced and band broadening will occur due to diffusion.” So now we’re trying to run our gels at higher voltages (90-120 V) for longer increments of time (3-4 hours). We haven’t quite exactly pinpointed these conditions, but as we continue on with this project we hope to discover the “ideal” setting.

I’m so glad I have been able to continue my research with Dr. Brokaw this summer. I feel like I’m finally getting comfortable around the lab–with the equipment, methods, and the people even. It was nice to be able to focus solely on research, which gave me a more realistic picture of grad school and the research field as a whole. This summer I encountered difficulties and made mistakes, but I also found successes and became more confident in my own abilities. It seems bittersweet to think that my summer work is coming to a close, but I am definitely excited to move forward with this project during the semester (preview of coming attractions: we will use primer pair 3 to identify many samples)!

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