Cats and Korlević

Doing research involves a lot more than just lab work. We also read a lot of papers from people who have done foundational research that we’re building from. Two such papers are “Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA” by Gansauge and Meyer (2013), and “Reducing microbial and human contamination in DNA extractions from ancient bones and teeth” by Korlević et al. (2015).

Gansauge and Meyer introduce and test a new method for improving the recovery of ancient DNA for DNA sequencing. In this method we take ancient DNA and add little bits of known DNA sequence to them that we can use later to copy the ancient DNA and sequence it. Many copies of the ancient DNA (with attached known sequences) are called a DNA library. In ancient DNA, many strands of DNA are often damaged and broken. DNA is present as two attached copies. Previous methods required both strands to be present and intact, but if they’re broken they get lost during processing. The thing that differs in this method versus others before it is that this method uses single-stranded DNA instead of double-stranded, so much more of the DNA is sequenceable. Another cool thing about this method is that they bind the DNA to magnetic beads so that they can be sure they aren’t losing any of it.

Korlević et al. talk about the contamination present in ancient DNA and how to decrease it. We don’t often think about it, but literally everything is covered with bacteria, and bacteria all have their own DNA. This means that any sample will be contaminated with bacterial DNA, and often with human DNA from handling. Korlevic et al. test three different methods of getting rid of the extra DNA: (1) using bleach on the samples, (2) incubating the samples in a phosphate buffer, and (3) re-extracting the DNA from the sample multiple times. Bleach was the best at getting rid of the contaminating DNA, but it also got rid of a lot of the target DNA, so it isn’t a good method for rare or precious samples. The phosphate treatment turned out to be the safest, since it preserved all of the target DNA, but it also didn’t get rid of as much contaminating DNA. The re-extraction method didn’t help significantly, and even made the contamination worse in some cases.

Reading papers like these is important for understanding the work that came before ours, and putting our own work into context. The papers can be difficult to read and understand, but reading them is an essential part of the research process. It’s also not too bad when you get to pet kitties while reading!

 

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Maren and me at Heather’s house reading papers with her cat, Legolas.

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Playing with fun machines

 

On Wednesday Chris Smith kindly showed us how to use fluorescence to determine how much mitochondrial DNA we amplified last week. This is done on a Quantitative PCR (qPCR) machine using picogreen.

When we ran our reaction, both long products and unintended smaller pieces amplified. We needed to separate the unwanted smaller products out from the longer ones. To run all nine samples using four primer pairs, we had to run two gels. Gels use charge to push DNA through the gel. DNA is negatively charged, so applying a negative charge forces the DNA to move through the gel. Bigger pieces of DNA will get stopped in the gel earlier and smaller pieces will travel further, so when looking at the gel after it is finished running, we can determine which bands are bigger pieces of DNA.

I enjoy making and running gels because we can finally see what we are dealing with. We mixed 1 g of Agarose in TAE, and then heated the mixture up in a microwave. Apparently Agarose is quite viscous and will burn you and refuse to come off if it touches your skin, so Heather kindly didn’t let us handle it while it’s heating. After the Agarose dissolved we poured the gels and learned the hard way to not assume that they’re set before trying to yank them up.

Unlike yesterday, we ran the gels at the recommended 60 mV so that our bigger chunks of DNA wouldn’t be pushed through the gel too quickly or get heated up and damaged. After an hour we read the gels under UV light and they both showed DNA, which was a good sign.

However, we still needed to get the DNA out of the gels. The Gel-Doc has a UV light underneath the tray that the gel sits on, so the DNA bands are illuminated. We cut the brightest bands (the DNA) out with a razor and put them in a tube. This was dicey because we had to wear sunglasses and a scratched or damaged UV shield so that the UV light didn’t burn us up. This made spatial reasoning more difficult. The bands were completely surrounded by the gel, so we had to cut the gel away from all six sides of the band while moving quickly enough so that the DNA wouldn’t be damaged by the UV light. While one person cut out a band, another would tare a labeled tube and then hold it out for the cutting person to put the band in, then weigh and record the tube, and finally return for another band and repeat the process. We had an exchange of tare puns with Peter’s group. There were some terrible ones and we finally had to tear ourselves away.

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Cutting out the bigger bands from the gel using the guidance of the Gel-Doc’s inner UV light. 

After we got the bands in the tubes with nobody burned from the UV light, we added a Guanidium buffer (more Guanidium! Yay!) to each tube and centrifuged the tubes. We ended the day by eluting the DNA cut from the gels and loading the qPCR machine. Unfortunately, we failed to retrieve the data but the next morning Chris saved the day and exported our data. Luckily the data was there; we had just messed up getting it off the computer. Unfortunately, the data wasn’t great: some of the samples that should have had similar values were drastically different and our standard curve was not a nice line. We figured that our pipettes were off and got permission to use Chris’s pipettes for the next time. We re-ran the picogreen assay and got improved results: our standard curve had an r^2 of 0.89 and the values were more similar across primers. Primers A-D had reasonable amounts of DNA but Primers E-H had extremely low amounts.

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Plastic knuckles for ensuring that the caps on the tubes are secure for the picogreen assay! 

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In which we calculate Molarity and get our First Results

The past few days have gone by quickly! Apparently, we did six days of work in our first five-day week, which was satisfying to learn. By the end of last week we had made our binding buffers, added them to our samples, and centrifuged them, and set up our Long Range PCRs.

However, Friday was the 13th, so some mishaps were to be expected. The Binding Buffer called for 39.8 g of Guanidine, a solid that looks like sugar that’s been sitting in someone’s china cabinet for 10 years. Guanidine becomes Guanidium in water, although it is notoriously hard to dissolve. Even still, 39.8 g seemed like too much, so we ran around and did math until we figured out that it was supposed to be 23.8825 g. Today, Heather found out that the protocol we had been using had miscalculations, and that we were correct to use 32.8825 g of Guanidine. That was a relief!

We have a vortex in the Ancient lab, but it strangely didn’t come with a plug. The cord just ended in a series of wires. Heather bought a plug and we wired it ourselves to no avail. In the end, we used our inner human vortexes and it was fine. At the same time, our pH meter didn’t want to calibrate, so Emily spent a long time fiddling with it. We needed to get the pH of the sodium acetate to 5.2. Eventually, we got close enough.

On Monday, we had a shorter day. In the morning, we made several Wash Buffers in the Ancient lab. Everything went smoothly. The only hiccup was when we discovered our SDS was 10% instead of 20%, so we compensated by using twice as much and adjusting the amount of water added to the solution. After lunch, we went up to the modern lab and made a gel to run the Long Range PCRs on. Previously, I had only used pre-cast gels, so it was satisfying to make the gel ourselves. We pipetted our DNA into the wells as quickly as possible so that the DNA couldn’t leak out. After the gel was finished running, we looked it on an imager. The gel (below) showed excellent amplification of partial mitochondrial genomes for the modern beaver!

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Gel of Beaver Mitochondrial DNA: There are two rows. The two long smears on each end of the top row and in the second to last lane of the bottom row show a DNA ladder with known (short!) sizes of DNA fragment. The other bands on the top row are ~11,500 bps of DNA, while the bottom row has ~6,000bp fragments of DNA. 

Now we can go forward turning those sequences into baits to fish out ancient DNA.

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A Beaver Up Your Sleeve, or What Happened on Wednesday

On Wednesday, we went into the Joseph Moore Museum research collections and decided on three museum specimens to sample for sequencing.

The first was a male Castor canadensis, the North American beaver. It was collected in 1965 from Beaver Creek in Alaska. Initially, we wanted to sample from one of its toe pads, but it turns out whoever prepped its skin turned its feet inside out so the pads were unreachable. Heather tried really hard to get to them, though!

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A beaver up your sleeve? Heather tries to reach the beaver’s toe pads.

 

We ended up taking a molar from its skull instead.

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Maren extracts a molar from the beaver specimen.

 

The second specimen was a male Geomys bursarius, the plains pocket gopher. It was collected in 2001 from Gimlet Lake in Nebraska. Pocket gophers are pretty small, and therefore don’t have very large toe pads, so we elected to sample teeth from this specimen as well.

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Plains Pocket Gopher (Geomys bursarius)

 

The final specimen was a Dipodomys ordii, the Ord’s kangaroo rat. It was also collected from Gimlet Lake in Nebraska, in 2010. The sex of this specimen was unknown because the tag was marked with an ambiguous gender symbol. This specimen had no skull associated with it, so we were sampled a toe pad. However, its entire right front paw was barely hanging on. We decided to help it along the rest of the way, and took the entire paw.

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Ord’s Kangaroo Rat (Dipodomys ordii)

In addition to sampling museum specimens, we also took time on Wednesday to clean the ancient lab. It had been a while since anyone had used it extensively, so it needed a deep clean. Everything, and I mean everything, had to be wiped down with bleach to get rid of any potential contaminating DNA. Benchtops, equipment, bags of tubes, and boxes of gloves, all bleached thoroughly. We swept the floors and organized everything as well, all while wearing full body suits and masks to prevent our own DNA from contaminating the lab. It was hard work, but everything looked great once we were done.

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All suited up for the ancient DNA lab (Left to right: Heather Lerner, Emily Buttrum, Maren Schroeder)

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Emily’s Introduction

Hi guys! My name is Emily Buttrum, and I’m Heather’s other research assistant for this summer. I’m a senior biology major from Indianapolis, and I have an intense love for all critters, giant and otherwise.

On Tuesday, we finished our extraction of the modern beaver DNA while practicing our pipetting skills. Micropipettes are surprisingly sophisticated and delicate tools used to transfer extremely precise volumes of liquids. Knowing how to use them properly is key to getting good results. Thankfully, good results are exactly what we got. A quick analysis of our extract with the Nanodrop system showed that it contained tons of useable modern DNA (more than 60 ng/ul!). We stored the DNA in the refrigerator until we get some of the materials necessary for the next step in the process: DNA amplification via PCR.

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Maren (left) and me, Emily (right), finishing the extraction of modern beaver DNA.

In the afternoon, we read a paper about the mitochondrial genome of the modern beaver and how slowly that DNA changes. In short, the study asserts that because beavers have such long lifespans (for rodents), their mitochondrial DNA changes very slowly. We think that this slow evolution could be a contributing factor to why every other past beaver species (38 in all!) have all become extinct.

In the process of reading the paper and filling out a “message box” on it, we also discovered that modern beavers might be most closely related to a family of rodents called Geomyoidea, which includes pocket gophers and kangaroo mice, but that has not been tested with mitochondrial genome data.

We want to sequence a genome from a specimen or two of Geomyoidea, to test this hypothesis.

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The Plains Pocket Gopher  (Geomys bursarius) is a member of the Geoymoidea.

 

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Ancient DNA Summer Research Intro

My name is Maren Schroeder, and I’m a junior at Earlham. I’m lucky enough to be one of Heather’s research assistants this summer. I’m a neuroscience and comparative languages and linguistics double major. In my spare time, I enjoy horseback riding, wandering around outside, petting the barn cats, eating peppers, and thinking. I’m excited to work with ancient Giant Beaver DNA because I’ve never done anything like it before and I love learning about DNA. No matter how this project turns out, I’ll learn something and that’ll be cool.

As for the actual project, our science-y goal is to extract DNA from an ancient Giant Beaver (Castoroides ohioensis). If you’ve been to the Joseph Moore Museum or follow our blog, you probably already know that Giant Beavers are an extinct species of beavers that lived during the Pleistocene era. We aim to see how genetically similar the Giant Beaver is to the modern beaver.

Since we may or may not be successful in extracting Ancient Beaver DNA, we made some other goals. Our other goals are to do science for ourselves and not for our grades, improve our lab skills, understand the nuances of working with ancient DNA, maybe publish something, and have fun. If we fail to get a single base pair of ancient DNA, that failure will be purely a learning experience and not a GPA-tarnishing humiliation. We’ll all learn something and it will be fine.

On our first day, we did a mishmash of everything, from meeting each other, going over our goals, writing a schedule that was erased and rewritten multiple times, ordering oligonucleotides (short DNA sequences that will be attached to the ancient DNA), finding some modern beaver tissue to get DNA from, digesting the tissue, and leaving it in a tube to float in some hot water overnight.

As it turns out, it is very unlikely to successfully extract and analyze ancient DNA. DNA degrades and makes errors over time. Living organisms have cellular mechanisms to catch errors in DNA replication, which is why we’re still alive. However, dead things do not have this ability, so their DNA basically sits and messes itself up for thousands of years before we come along and try to analyze it. The DNA may be mixed with bacterial DNA from the outside environment, various proteins may chop the DNA up, there might be a T base pair where there was originally a C base pair, heat may degrade the DNA, and the list goes on. Therefore, any ancient DNA strands we get will be very short, likely less than 100bp. Depending on the environment, DNA that is 100,000-1,000,000 may be preserved. DNA will be better preserved in cold, dry environments.

After going over all of that, we got to work. We found some modern beaver tissue in a freezer, weighed it (about 21 mg), chopped it up into tiny pieces and put the pieces into a tube, added some enzymes, and left the tube to sit in a hot water bath overnight.

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Emily weighing out modern beaver tissue.

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Getting tiny bits of chopped up beaver tissue in a tube.

We read a paper about the evolution of swimming and tree-exploitation behavior in beavers, which found that swimming and woodcutting evolved in beavers only once, and had implications for how beavers might evolve/react to climate change in the future. The paper noted that beavers survive winter by creating food caches underwater from trees that they cut down themselves. The chemical defenses in Poplar trees probably evolved as defenses against beavers. If the earth becomes warmer and beavers stop creating caches, there could be secondary effects on the species that interact with beavers, including animals that live in the lakes created by beavers and the trees beavers predate.

 

That’s all for now! Thanks for reading!

Maren

 

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Spring Break Adventure in Ann Arbor, MI

What do JMM staffers do for spring break? Explore museum collections and attend ecology symposiums of course! And what better place than the University of Michigan in Ann Arbor? JMM Museum Director Heather Lerner and eight students set off early Friday morning March 10th to Ann Arbor.  After arriving at the Natural History Museum at the University of Michigan, we explored the exhibits–including a special exhibit on evolution! Soon after, we met up with Kirra Berman, Associate Director of Education, and John Klausmeyer, Exhibit Preparator.  They showed us around the museum’s Hall of Evolution, which is filled with fossils, dinosaur fossils, mastodons, and other remains of fascinating creatures (although no  complete Giant Beavers!).

Two mastodon skeletons in the  “Hall of Evolution” (Photo:www.lsa.umich.edu/ummnh)

We also talked about the Natural History Museum’s new home. Don’t worry, the move isn’t far–across the street is the site of the university’s new Biology Building where the museum will find a more spacious and modern abode. While some popular exhibits will remain, most of them will be redesigned to fit the new space, John told us.

Next, we met with graduate admissions directors Trish Witkopp and Cindy Carlson who offered tips and advice to our students on finding graduate programs and being accepted.

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Two birds of the collections

Finally, we visited the vast museum collections. Earlham College alum Janet Hinshaw showed us the Bird collection, which houses 6,387 species, two thirds of the species in the world! We saw a wide array of species, including kiwis, rheas (the only bird with a bladder) and ivory billed woodpeckers, an extinct species.

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Julie Anderson holds an Ivory Billed Woodpecker

Then on to the Insect collection where we saw everything from luna moths, to hawk moths, to an orchid mantis. Can you guess what this large insect with the colorful wing is? 

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It’s a grasshopper!

Afterward, we finished off the afternoon with a delicious dinner at Zingerman’s Delicatessen. We enjoyed sandwiches, cheese, and good conversation with two U of M graduate students and Dr. Robyn Burnam, who told us about her research on vines in the Amazon Rainforest.

The next morning found us munching on bagels and bananas while waiting for the start of the 12th Annual University of Michigan Early Career Scientists Symposium. This year’s theme was “Frontiers in Community Assembly.” University of California, Berkeley Professor Rosemary Gillespie gave a fascinating talk on spider evolution on the Hawaiian Islands. We also heard about island lizard evolutions, fungi, and what the fossil record can reveal about community assembly!

The Hands On Museum focuses on interactive exhibits that are fun for kids and adults. (Photo:www.aahom.org)

On Sunday, a handful of us visited the Hands on Museum, where director of education Laurrie Beaumont gave us a behind the scenes tour. She explained two exciting new programs at the museum, sensory kits and distance learning. Sensory kits are kits of sunglasses and ear protectors which help visitors who can become overwhelmed by the highly stimulating environment at the Hands On Museum. Using these kits allows visitors to be more comfortable and better enjoy their museum experience. Distance learning allows classrooms that can’t travel to a museum the opportunity to connect with museum educators virtually. The museum provides the class with an activity kit and then Skypes with the class to guide them through the activity. The Joseph Moore Museum is currently piloting similar activity kits to be used in the museum and in the classroom, so stay tuned!

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Richmond is a time, too!

Richmond is our town, but did you know that it’s given its name to a section of geological time? The Richmondian age lasted from about 449 to about 445 million years ago, during the Ordovician Period.

 

Our town looked very different 449 million years ago! It was so close to the equator that it was warm and humid. It was at the bottom of a shallow sea that covered most of what is now the midwestern United States. There were large mountains to the east, with volcanoes like those now found in Japan.  These types of volcanoes are formed as an ocean plate subducts, or moves underneath, a continental plate.  When the ocean plate moves down into the Earth’s mantle and melts, the magma rises through the continental plate to form volcanoes.

 

The Richmondian ocean was full of life, but we would not recognize most of the creatures living there, such as shelled animals called brachiopods (Greek for “arm-foot”).  Even though brachiopods are related to mollusks like clams or scallops, their bodies are quite different: clams’ shells are symmetrical and they have a left and right shell that are shaped the same, but brachiopods have a top and bottom shell that are shaped differently.  The rocks around modern Richmond have fossils from many different brachiopod species, including  Hebertella, Hiscobecus, and Rafenesquina.

 

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Hebertella fossil. Source: Wikimedia contributor dwergenpaarje.

 

Corals, bryozoans, and crinoids were also common in the Richmondian ocean.  Corals first became common in this area during the Richmondian.  Most of the coral reefs in today’s oceans are formed by many individual corals (a relative of jellyfish!) living in colonies together.  The Richmondian horn corals were solitary, with “horn” the home of a single animal.  The Richmond Fossil Park contains many fossils of the horn coral Grewingkia.

 

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Grewingkia fossil. Source: Wikimedia contributor Wilson44691.

 

One animal that looked very similar to today’s colonial corals, and their fossils have many small openings where the individual animals lived were the bryozoans (“moss-animals”). Bryozoans may look a lot like corals, but they are actually more closely related to brachiopods.

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Bryozoan fossil. Source: Wikimedia contributor Regiomontanus.

The strangest looking of the Richmondian’s ocean creatures – they looked like an upside-down starfish on a stem – are the Crinoids (“lily-forms”). The stem kept their arms off the bottom so that they could catch food from the water.  Crinoid stems are very common in the rocks of modern Richmond.

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Crinoid fossil. Source: Wikimedia contributor Vassil.

The top predators of the Richmondian ocean were nautiloids, distant relatives of today’s squids.  They had shells that looked like torpedo cases and could be anywhere from a few inches to eight feet long.  A hungry nautiloid could eat anything it wanted!

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A artist idea of what a nautiloid looked like. Source: Wikimedia contributor Nobu Tamura. 

The astonishing animals that lived here so long ago are called the Richmondian Fauna, and their fossils are very common in the rocks around modern Richmond.. To learn more about nearby fossil-hunting sites, visit the Joseph Moore Museum and get your fossil passport!

 

Sources:

https://www.tonmo.com/pages/nautiloids/

http://www.indiana.edu/~paleoind/Lectures/Lecture%209%20-%20Ordovician%20and%20Richmondian%20Invasion.pdf

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Wrapping Up The Semester: Overview and Accomplishments

The museum has had a productive and busy semester. Our collective efforts have allowed us to put on successful museum events, improve the museum exhibits and programs, and reach out to the community as a whole.

 

During this Fall Semester, we have accomplished the following:

 

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A reusable display was made for traveling advertisement and for events in Richmond. This new display debuted at an event in the park in the Richmond community. We had great help from graphic designer, Noah Medlen, who taught us about branding, visual communication, and marketing. A photo-booth Allosaurus and Triceratops head was also made in the wood shop for such occasions, so people may take dinosaur selfies. In addition, an Allosaurus claw was made by the 3D printer and is now used by hosts of the museum to give visitors a closer, more hands-on look at the Dinosaur that we have.

 

 

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The new CST building at Earlham has allowed us to put up a display in order to show the public about what we have to offer! An informational poster and artifacts from our collections were picked and placed into the display case. The CST display was done and put up in time for Homecoming and received a good amount of positive feedback.

 

 

 

 

 

 

 

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We talked with authors of a new study about the track patterns of Mastodons, and found that the size, shape, and arrangement needed to be altered in order to be scientifically accurate. Therefore, Fab Lab measured and cut new stencils in order to re-paint them. New Allosaurus footprints were painted over the old, fading ones. Follow these tracks across campus to give us a visit!

 

 

 

 

 

 

 

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The Climate Change event allowed the museum to unveil a new exhibit, and it was a huge success! New photographs were hung that had been gathered from public and professional submissions. The opening of the exhibit featured many interactive stations to help the visitors better understand climate change. These stations ranged from screen-printing to investigating climate data through technology. We are so grateful for everyone’s collective efforts and participation. A big thanks to all museum staff, the class Environment and Society taught by Jamey Pavey, the Science and Pseudoscience class taught by Michael Lerner, CS Applied Groups, Cope Environmental Center, and the Richmond Art Museum. Over 200 people attended this event!

 

 

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One of our Fab Lab members cut and painted 60 blocks in order to make Ecosystem Jenga! This game was a huge success at the Climate Change event. It serves to be a fun and interactive activity, while also educating the participants about the effects on ecosystems and the interactions within them. Jenga will be used in future events as well, so look forward for some more toppling fun!

 

 

 

 

 

 

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Fab Lab is working on creating a new logo for the Joseph Moore Museum, and has been brainstorming on and reviewing many different ideas. This logo will go on buttons and other paraphernalia for the promotion of the museum. The Giant Beaver is the new approach for representation for the museum.

 

 

 

The overall goals of this semester were to create new programs and activities, improve on what we have to offer, and to bring the Joseph Moore Museum closer to the Richmond community and to the students of Earlham College. As a museum team as a whole, we feel that we have had a successful and engaging semester, and we are proud of all that we have accomplished.

http://www.jbc.org/content/259/1/224.full.pdf

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Homecoming Weekend

This past weekend, the museum held a Homecoming Alumni Open House from 10 a.m. until 2 p.m. The event was a smashing success, with over 150 visitors stopping by the museum, some of whom increased our Memory Jar collection, leaving behind memories of their time here at the Joseph Moore Museum. Visitors also got to enjoy a sneak peak at our new exhibit on climate change. Alumni, current students, and faculty roamed the entire museum, commenting on the changes that have taken place, the climate change sneak preview, and enjoying refreshments in the lobby while chatting and sharing memories. Having the chance to talk with past museum staff members provided our current staff members with new tips and tricks and connected past generations to current members.

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