In Vitro Transcription: Common Causes of Reaction Failure

Posted on by Kelly Grooms
FemaleWhiteLab-AAES001042, In Vitro Transcription

A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies (X-ray crystallography and NMR). Synthesized RNA transcripts are also used for studying cellular RNA functionality in processes such as splicing, RNA processing, intracellular transport, viral infectivity and translation.

Problems in the transcription reaction can result in complete failure (i.e., no transcript generated) or in transcripts that are the incorrect size (i.e., shorter or longer than expected). Below is a discussion of the most common causes of in vitro transcription problems.

Continue reading “In Vitro Transcription: Common Causes of Reaction Failure”

How Do You Solve a Problem Like Malaria?

Posted on by Mariel Mohns
malaria_researcher
Photo courtesy of NIH/NIAID

Malaria affects nearly half of the world’s population, with almost 80% of cases in sub-Saharan Africa and India. While there have been many strides in education and prevention campaigns over the last 30 years, there were over 200 million cases documented in 2017 with over 400,000 deaths, and the majority were young children. Despite being preventable and treatable, malaria continues to thrive in areas that are high risk for transmission. Recently, clinicians started rolling out use of the first approved vaccine, though clinical trials showed it is only about 30% effective. Meanwhile, researchers must continue to focus on innovative efforts to improve diagnostics, treatment and prevention to reduce the burden in these areas.

Continue reading “How Do You Solve a Problem Like Malaria?”

Lab Sustainability Doesn’t Have To Be Painful

Posted on by Jordan Villanueva

Ian Nicastro says he didn’t set out to start a green revolution.

“I’m not hardcore ‘Save the trees,’” Ian says. “I’m probably a little different from the people you traditionally see as promoting the sustainability thing. Obviously, I do want to help the environment, but for me it was like, ‘this is logical, and we should be doing this.’”

Ian is the lab manager of the Pasquinelli Lab, a C. elegans lab at the University of California–San Diego that studies miRNA and its role in processes like aging. He’s been in the lab for about six and a half years, splitting his time between research and lab management duties. According to Allison Paradise, the CEO of My Green Lab, Ian has put out some “outstanding” efforts to implement sustainable practices in the lab. Continue reading “Lab Sustainability Doesn’t Have To Be Painful”

Characterizing Compound Binding in Cell-Free Systems

Posted on by Promega

Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).

In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.

The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).

cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System  to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.

The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.

Reference
Kim, E-Y (2019) The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos). Ecotoxicology and Environmental Safety 171. 99–111

Are We Doing Enough to Stop Candida Auris Infections?

Posted on by Kari Kenefick
Image of C. auris on plate.
The creamy colonies of C. auris look innocuous. Don’t be fooled. Photo by Shawn Lockhart – Centers for Disease Control, Public Domain, https://commons.wikimedia.org/w/index.php?curid=54680002

Life in the 21st century is full of electronic devices and apps purported to make life easier. Many of us can binge watch movies, videos and news on our phones. There are wireless headphones, electric bicycles, self-operating vacuum cleaners, wine in boxes with taps—and so much more.

This life is, however, not without challenges.

In the event that you or yours ends up in the hospital, the stay could be complicated by an unplanned, unwanted and potentially lethal infection.

No thanks to the yeast, Candida auris.

Continue reading “Are We Doing Enough to Stop Candida Auris Infections?”

Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?

Posted on by Terri Sundquist
Confused woman

I’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!

Sound familiar? Not to worry! The choice is not difficult once you know how these assays work and how they differ.

Continue reading “Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?”

Ocean Acidification Turns Sea Cucumbers into Sea Pickles

Posted on by Jordan Villanueva

A startling report from researchers in Washington indicates that the increasingly acidic waters of the United States Pacific Northwest are turning sea cucumbers into sea pickles.

Continue reading “Ocean Acidification Turns Sea Cucumbers into Sea Pickles”

For the Birds: Knitting Nests for Baby Birds Might Just Help Your Health To

Posted on by Kelly Grooms

It seems that spring has finally come to Southern Wisconsin. The snow has melted. Most days it is warm enough you can go outside without a parka, hat and mittens. The tree buds are starting to swell. And that traditional oracle of spring, the American robin (Turdus migratorius), has been spotted in trees and yards—along with its less friendly cousin, the red winged black bird (Agelaius phoeniceus).

While spring brings the return of migratory birds, it also brings an increase in the number of rescued baby birds flooding into local wildlife rescues and humane societies. When the babies come to these centers, they need a warm, soft, breathable and washable home that resembles the nest they were hatched in.

It turns out that knitted or crocheted nests are a perfect solution. The nests aren’t just used for baby birds; baby rabbits, squirrels, bats, ferrets and racoons are just a few additional animals that benefit. And the best part is, you could be improving your own health while you create those cozy nests. Continue reading “For the Birds: Knitting Nests for Baby Birds Might Just Help Your Health To”

When Good Proteins Go Bad

Posted on by Natalie Larsen

Ribbon model of p53 protein bound to DNA molecule.
Ribbon model of p53 protein bound to DNA molecule.

Following what feels like an exceptionally long and brutal winter, I for one couldn’t be happier about the arrival of Spring and the way it makes everything seem brighter and brand-new. Soaking in the soul-warming sunshine. Reveling in the sweet melody of chirping birds. Watching the earth literally coming alive again with greenery. And for those of us who love and are enthralled by scientific discoveries like myself, the report of a recent shiny new discovery in the world of cancer research is equally as day-brightening and spirit-lifting.

To suppress tumors or to not suppress tumors: that is the question.

In the world of oncology, the protein known as p53 has long proven itself to be a primary target of interest. p53 operates as a tumor suppressor protein, often lauded as the “guardian of the human genome”, due to its dedication to governing controlled cell division and assessing damaged DNA. There are a number of cellular stressors that can wreak havoc on your DNA, including exposure to ultraviolet light or radiation, oxygen deficiency (hypoxia), and contact with hazardous chemicals.

Consider a normal-functioning p53 protein as the quality control person in a production factory. The p53 protein evaluates the products, DNA, coming down the line and determines an appropriate course of action for those that do not meet the quality standards.

Let’s say some less-than-quality DNA comes down the pipe. If the DNA is not too severely injured, p53 will alert and activate additional genes to repair the damage. However, if the products coming through are too marred to repair, p53 will shut down the whole factory, if you will, by signaling for the cell to self-destruct via apoptosis. In doing so, p53 effectively impedes tumor development by inhibiting the ability for flawed DNA to further divide.

So, it would seem like p53 has proven itself to be an undeniably upstanding citizen of the protein variety, right? The unfortunate truth of the matter is p53 balances delicately on a double-edged sword, establishing itself as the veritable Dr. Jekyll and Mr. Hyde of the cellular world: usually unquestionably good, but sometimes unspeakably evil. Continue reading “When Good Proteins Go Bad”

Cloning Modified Blunt-ended DNA Fragments into T-Vectors

Posted on by Kelly Grooms

Tailing blunt-ended DNA fragments with TaqDNA Polymerase allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors. This method also eliminates some of the requirements of conventional blunt-end cloning — Fewer steps, who can argue with that?

Blue/White colony screening helps you pick only the colonies that have your insert.
Blue/White colony screening helps you pick only the colonies that have your insert.

Continue reading “Cloning Modified Blunt-ended DNA Fragments into T-Vectors”