Neuroscience Explains Harry Potter’s Appeal

Posted on by Terri Sundquist

BookWithGlassesCurling up with a good book is one of life’s greatest pleasures, whether you’re reading on a tropical beach while on vacation or nestled into your favorite chair at home. As your eyes skim over the words, your mind conjures up images of the events unfolding on the page. Books can take us to fantastic places, real and imaginary, that we will never visit in our lifetime. And while there is some pleasure to be gained from nonfictional books, my favorite books all seem to fall in the realm of fiction. I am not alone. The science fiction and fantasy genre of literature continues to be one of the most popular. Why do so many readers find these types of books so enticing and engaging?

It all comes down to science, specifically neuroscience.

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Culturing the Unculturable Bacteria

Posted on by Isobel Maciver
Culturing bacteria is't always this easy.
Culturing bacteria is’t always this easy.

It is estimated that all the bacterial species so far described represent only a tiny fraction of the total. The rest remain unknown to science because they are “unculturable” in standard (or known) laboratory media. Given that many antibiotics were first isolated from environmental bacteria, it seems likely that these as yet unknown organisms could also be a rich source of potential new drug candidates. The desperate need for new strategies to combat multi-drug resistant infections gives impetus to studies investigating how we can culture some of these “unculturable” bacteria and uncover their potential as a source of much-needed new treatments. 

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Announcing the 10th Annual Wisconsin Stem Cell Symposium: Engineering Limb Regeneration

Posted on by Karin Borgh

Image courtesy of James Monaghan, Ph.D., Northeastern University, Boston, MA
Image courtesy of James Monaghan, Ph.D., Northeastern University, Boston, MA

On April 22, 2015, the BTC Institute and Promega Corporation will host the 10th Annual Wisconsin Stem Cell Symposium — Engineering Limb Regeneration: Recapitulating Normal Development and Regeneration? Our colleagues at the University of Wisconsin-Madison have put together an outstanding list of presenters who will address advances – and challenges – associated with this field of research.
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Mitochondrial DNA Typing in Forensics

Posted on by Anupama Gopalakrishnan

23540687_lMitochondria, often thought of as powerhouses of the cell, are fascinating eukaryotic organelles with a double-layered membrane and their own genome. Mitochondrial DNA (Mt DNA) is typically about 16570 bases, circular, highly compact, haploid and contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation, a process that uses oxygen and simple sugars to create adenosine triphosphate (ATP), the cell’s main energy currency. The remaining genes code for transfer RNA (tRNA) and ribosomal RNA (rRNA) which are necessary for translating messenger RNA transcribed from nuclear DNA, into protein molecules.

One of the most important characteristics of mitochondrial genome that is relevant to field of forensics is the copy number. Continue reading “Mitochondrial DNA Typing in Forensics”

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.

Friday Fun: A Cartoon Limerick for the Poetic Scientist

Posted on by Promega

Your Promega Connections bloggers thought we would inject a little levity into the day. So we are sharing this Ed Himelblau cartoon limerick.  Are any of our readers poets? Do you have a couplet, haiku or science-related limerick you can share with us?

eh16_maize

Tracking the Beginning of a Pathogenic Bacterial Infection

Posted on by Sara Klink

Yersinia pestis by U.S. Center for Disease Control [Public domain], via Wikimedia Commons.
Understanding the course of a pathogenic infection involves not only understanding what ultimately kills the host or how the bacterium or virus enters the body but also how it establishes itself in the host organism. What is the receptor that allows a virus to enter the cell? Which cells does a bacterium first target or how does it evade an immune response? While other studies of bacteria like Yersina pestis have looked at imaging the bacterial burden in model mice, questions remain about how this bacterium gets from the skin after an infected flea bites to the draining lymph nodes, where the bacteria replicate and enter the bloodstream and infection becomes fatal. A recent PLOS Pathogens article examined how the nonmotile Y. pestis disseminated itself starting from a tiny innoculation mimicking a flea bite on a mouse ear and following pathogen interaction with the host from skin to lymph node. Continue reading “Tracking the Beginning of a Pathogenic Bacterial Infection”

Choosing the Best Luciferase Vector for Your Experiment—Now Made Easier with the Vector Selector

Posted on by Michele Arduengo, PhD

4621CAGenetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).

Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation

However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.

At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?

To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.

So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.

How Confident are You in Your Mass Spectrometry Data?

Posted on by Gary Kobs

Data generated by scientific instruments and decisions based on that data depend on optimal instrument performance. Clinical assays rely on mass spectrometric (MS) data for accurate results so that correct health related results are gained and appropriate results-based decisions are made. However, there are no generally agreed upon tools nor performance standards for mass spectrometry. Furthermore, while several software tools exist that serve to assist  with the analysis of instrument performance, a dedicated reagent software package has yet to be created. For optimal liquid chromatography (LC) performance, parameters like retention time, peak width and peak heightare typically reported. Commonly monitored MS parameters include mass accuracy, mass resolution, signal-to noise, sensitivity, limit of detection (LOD), limit of quantitation (LOQ) and dynamic range.

The 6 × 5 LC-MS/MS Peptide Reference Mix is designed for use in method development and optimization,and for routine liquid chromatography (LC) and mass spectrometry (MS) instrument performance monitoring. The product is a mixture of 30 peptides: 6 sets of 5 isotopologues of the same peptide sequence. The isotopologues (Figure 1) differ only by the number of stable, heavy-labeled amino acids incorporated into the sequence. The labeled amino acids consist of uniform 13C and 15N atoms. Each of the isotopologues is indistinguishable chemically and chromatographically. However, since they differ in mass, they are clearly resolved by mass spectrometry.

Figure 1.
Figure 1.

The isotopologues of each peptide are present in a series of tenfold differences in concentration or molar abundance. If 1pmol of the mixture is loaded onto an LC column, the next lighter isotopologue would be 100fmol, the next 10fmol, the second lightest 1fmol, and the lightest 100amol. This range allows assessment of the instrument’s dynamic range and sensitivity from a single run.

Peptides with a wide range of hydrophobicities were chosen to enable reporting of LC column performance. The most hydrophilic peptide gives users a tool to optimize the capture of hydrophilic peptides that might be difficult to capture otherwise, but that are too precious to use for method development.

To assist in data processing, a complementary software tool, is provided, the 6 × 5 LC-MS/MS Peptide Reference Mix Analysis Software (The PReMiS™ Software). The PReMiS™ Software produces a tabular report of calculated instrument parameters, graphical analysis of linearity curves as well as reporting the history of user-selected parameters such as LC retention time, peak height and mass accuracy. If the laboratory has a collection of instruments, there is also an option to compare parameters across instruments.

A Whole New Meaning to the Phrase “Catching Cold”

Posted on by Terri Sundquist

ThermometerEvery day we are bombarded by potential contagions: whether a physical ailment such as measles or influenza or something as seemingly harmless as a yawn or popular Internet meme. For better or worse, emotions can be contagious too, passed on from one person to another through verbal and nonverbal cues, with or without the awareness that we are being affected by another person’s emotional state. In many cases, the only route for such transmission is observation. For example, who hasn’t felt better after watching an uplifting film or cried while watching a sad movie. In the lab, scientists have determined that levels of the stress hormone cortisol increased in individuals undergoing a stress test but also in passive observers who watch the stress test through a one-way mirror or on a television screen. Often, the magnitude of the observer’s response is affected by how well he knows or can relate to the person.

As reported in a recent PLOS ONE article(1), we now know that even a physiological response to cold temperatures is readily transmissible from one person to another, although many of us who live in northern climates probably knew this long before any scientific study: Watching children playing in the snow or someone shoveling snow can often send a chill through us even though we are watching from a heated building. However, a group of scientists in the UK and Germany was not satisfied with anecdotal evidence of temperature contagion, as they named this phenomenon. They did the experiment and generated the data.

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