Author: Kelly Grooms

The Volcano Beneath our Feet

Posted on by Kelly Grooms
Photo credit: Kelly Grooms
Firehole River, Yellowstone National Park. Photo credit: Kelly Grooms

This summer my family and I vacationed on one of the world’s largest active volcanoes. We weren’t alone. Every year over three million people visit this super volcano.

Yellowstone National Park covers almost 3,500 square miles in the northwest corner of Wyoming (3% of the park is in Montana and 1% in Idaho). The park is famous for its hydrothermal features, including the Old Faithful Geyser and vivid hot springs such as the Grand Prismatic Spring.

The park’s hydrothermal system is the visible expression of the immense Yellowstone volcano; they would not exist without the underlying partially molten magma body that releases tremendous heat. National Park Service website

Photo Credit: Nathan Grooms
Grand Prismatic Spring, Yellowstone National Park. Photo Credit: Nathan Grooms

These features are all visible reminders of the immense volcano that exists beneath the surface.  Recently, a team of seismologists discovered a reservoir of partly molten rock 12–28 miles beneath Yellowstone National Park. This video from Science 360 describes the discovery and why scientists are interested in it. It is important to note that this discovery does not mean that there is new activity or that the volcano under the park is closer to erupting. It does mean that scientist now have a better picture of the underground “plumbing”.

Getting the Most DNA from Your Plasmid Prep

Posted on by Kelly Grooms

dificult_cloningPlasmid DNA preparation is one of the fundamental techniques of molecular biology research. It goes without saying that you want to maximize your plasmid DNA yield as much as possible. Below are a few tips to help you get the most out of every Plasmid prep.

Increase Your Culture Volume

Most plasmid prep systems can process a range of volumes. For example, the PureYield™ Miniprep System can process 600μl to 3 ml of culture. Note: Exceeding the recommended culture volume can result in decreased yields because the increased biomass can lead to insufficient lysis and lysate clearing problems.

Use Optimized Culture Conditions

Media: We recommend growing cells in 1X Luria-Bertani (LB) medium. The use of rich medium, such as 2X YT, CircleGROW®, Terrific broth, or LB-Miller medium, which contains more NaCl, can significantly increase yields, provided that the biomass is within the acceptable range.

Culture incubation times: Low-density bacterial cultures yield relatively low amounts of DNA. Overgrown cultures produce suboptimal yields and excessive chromosomal DNA contamination. Do not use cultures grown longer than 18–20 hours. Continue reading “Getting the Most DNA from Your Plasmid Prep”

A Warm Body in the Mesopelagic: The Endothermic Moonfish

Posted on by Kelly Grooms

If you think back to biology class, you most likely learned that endothermy, or the ability to maintain a body favorable body temperature (i.e., different than the ambient temperature), was a unique characteristic of mammals and birds. This ability sets “warm blooded” animals apart from “cold blooded” ectotherms such as reptiles and fish.

The warm blooded opah
The warm blooded opah. Image from:USA NOAA Fisheries Southwest Fisheries Science Center. https://swfsc.noaa.gov/ImageGallery/Default.aspx?moid=4724

For fish, one of the biggest challenges to maintaining an elevated body temperature is convective heat loss in the gill lamellae.  There are a few fish species that are able to retain some of the heat generated internally, but these “regional endotherms” are only able to increase the temperature of specific areas or tissues.  Regional endotherms limit the heat loss with retia mirabilia, which are a complex network of blood vessels that act as counter-current heat exchangers that warm the cold arterial blood as it returns from the gills. However, these retia have only been found associated with specific muscle groups and organs, and as a result the rest of the fish’s body remains at ambient temperature.

A silver and crimson-colored, tire-sized, fish called the opah (Lampris guttatus), or moonfish, is changing what scientist thought they knew about endothermy in fish (1).  The opah lives in the cold, dim waters of the ocean’s mesopelagic zone hundreds of feet below the surface. Unlike other fish at these depths, the opah is a fast swimming, agile predator.  Its secret is retia located inside the gills. The location of the retia means that the opah can maintain an elevated body temperature throughout its body, and a warmer body temperature means it can swim faster and react more quickly than both its prey and other predators.

I don’t think that we should throw out all our biology text books and start rewriting descriptions of what defines a fish, but the discovery of the warm blooded opah should remind us that what we know is just a small drop compared to what remains to be discovered.

Reference

  1. Wenger, N. et al. (2015) Whole-body endothermy in a mesopelagic fish, the opah, Lampris guttatus. Science 348, 786–9.

Animal or Plant? FISH Labeling Reveals Horizontal Transfer of Algae Gene into Sea Slug Chromosome

Posted on by Kelly Grooms

There are times when I ask myself why I chose a career in science. This happens on what I call “grass is greener” days. On these days I dream of other careers—like National Geographic reporter or Caribbean tour guide–which all sound way more exciting than scientist. Admittedly these alternative careers are not ones that many people have the privilege of attaining, but sometimes reality gets to take a vacation. Fortunately, science is a fast-moving, always-changing field. As much as I might occasionally dream of exotic jobs in far away locations, science always pulls me back in with something new and unexpected. Because as much as we’d like to think we know, the truth is there is so much more that we don’t.

Image from: Pelletreau KN, Weber APM, Weber KL, Rumpho ME (2014) Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica. PLoS ONE 9(5): e97477. doi:10.1371/journal.pone.0097477
The sea slug Elysia chlorotica. Image from: Pelletreau K.N., et al. (2014)  PLoS ONE 9: e97477.

A case in point—sea slugs. These unfortunately named, exotic looking creatures have some surprising secrets. Continue reading “Animal or Plant? FISH Labeling Reveals Horizontal Transfer of Algae Gene into Sea Slug Chromosome”

DNA Purification from Plants: Not All Methods are Equal

Posted on by Kelly Grooms

pestle and mortar with leavesIsolating DNA from plant tissues is difficult for many reasons.  Unlike animal cells, plant cells have rigid cell walls, often made of tough fibrous material, and contain proteins and enzymes and other compounds such as polysaccharides and polyphenols that play a role in different cellular processes. These compounds can interfere with DNA isolation as well as downstream applications such as PCR.  For these reasons, DNA isolation methods that are used successfully for other sample types may not work well to isolate DNA from plant material. Continue reading “DNA Purification from Plants: Not All Methods are Equal”

PCR Cloning: Answers to Some Frequently Asked Questions

Posted on by Kelly Grooms

eh1Q: What is the easiest way to clone PCR Products?

A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.

Q: What if my DNA polymerase has 3´ to 5´ exonuclease activity (i.e., proofreading activity) that removes the 3´-end overhang?

A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.

Set up the following reaction in a thin-walled PCR tube:

1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl

Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.

Q: What is a T-vector, and why are they used for cloning PCR products?

A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.

For a complete PCR Cloning protocol, Visit the Cloning Chapter of the Promega Protocols and Applications Guide.

Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast

Posted on by Kelly Grooms

Starfish BlueA killer is lurking in the waters off the pacific coast. Silent and lethal, it leaves its decimated victims in tidal pools. They first began to appear in the early summer of 2013. Limp and curled, missing some or all of their limbs, the bodies were little more than globs of slimy tissue.  They were hardly recognizable as what they once were—Sea Stars. Continue reading “Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast”

When DNA Is Not Enough: New Research Suggests Epigenetic Factors Play an Important Role in the High Mortality Rate of the Devi Facial Tumor Disease

Posted on by Kelly Grooms

tazThe Devil Facial Tumor Disease (DFTD) is a contagious cancer in Tasmanian Devils that is threatening the species with extinction. This disease is spread from individual to individual and has a 100% mortality rate. It is so deadly because, although the DFTF cells should be attached and killed by the host devil’s immune system, for some reason they are not—and no one is sure why. A study published in PNAS in March of last year (1) showed that DFTD cells don’t express surface MHC molecules. MHC class I and class II molecules are crucial for proper immune response, and their absence on the cell surface could explain why the DFTD cells do not stimulate an immune response.

The authors found that the loss of MHC expression is maintained as the cells divide, and is not a result of structural mutations in the genes responsible for MHC expression. Instead the authors found that this down regulation was the result of regulatory changes including epigenetic modifications to histones. Continue reading “When DNA Is Not Enough: New Research Suggests Epigenetic Factors Play an Important Role in the High Mortality Rate of the Devi Facial Tumor Disease”

Choosing Your Subcloning Strategy

Posted on by Kelly Grooms

Before you begin your subcloning, you need to know: The restriction enzyme (RE) sites available for subcloning in your parent vector multiple cloning region (or in the insert if you need to digest the insert); the RE sites available in the destination vector multiple cloning region (MCR); and if these same sites also occur in your insert. Once you know this information, you can use the chart below to decide which subcloning strategy to use.

4498MA-[Converted]

To learn more about subcloning, visit our Subcloning Notebook.

RNAi: The Dream Makes a Comeback

Posted on by Kelly Grooms

This Promega Notes Cover from 2004 celebrated the potential stop and go power of DNA-directed RNAi.
This Promega Notes Cover from 2004 celebrated the potential stop and go power of DNA-directed RNAi.

In the early 2000s, RNAi was a hot topic. The science world was abuzz with all the possibilities that harnessing this natural process could hold. And why not? The idea of posttranscriptionally silencing genes using only a small fragment of double-stranded RNA is huge—big enough to earn the scientists who discovered it a Nobel Prize in 2006.

The process of RNAi starts with short (~70 nucleotieds), double-stranded fragments of RNA called short hairpin RNAs (shRNA). These shRNAs are exported into the cytoplasm and cleaved by the enzyme Dicer into smaller pieces of RNA that are about 21 nucleotides long and are referred to as small interfering RNAs (siRNA). The siRNAs reduce or stop expression of proteins through a sequence of events where the antisense strand of the siRNA is incorporated into and RNA-induced silencing complex (RISC), which then attaches to and degrades its complimentary messenger RNA, thereby reducing or completely stopping expression.

It turned out, however, that harnessing the promise of RNAi was a little trickier than anticipated. Continue reading “RNAi: The Dream Makes a Comeback”