Two-Sentence Scary Lab Stories

jackolantern_editYour bloggers at Promega Connections like Halloween. In the past we have reviewed our top scary blogs and provided lists of things to do with pumpkins and suggestions for what to do when you have too much leftover Halloween candy. This year we are jumping on the 2-sentence horror story bandwagon with a twist: the 2-sentence scary/funny lab story. Here are a few of our creations. If you have one of your own, leave it in the comments.

saveI had just finished writing the final chapter of my dissertation, when a pop up box appeared :“Are you sure you want to exit without saving changes”. Then the screen went blank. Continue reading

Genome Editing and Reporter Technologies Enable Endogenous Pathway High-Throughput Assays

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;



This article review was written by guest author, Amy Landreman, in the Cellular Analysis and Proteomics Group at Promega.

Charcot-Marie Tooth (CMT) disease is one of the most common inherited neurological disorders affecting approximately 2.8 million people worldwide. The most common form of CMT, CMT Type 1A, is caused by a 1.5Mb genomic duplication on Chr17 that results in trisomy of the critical myelin gene Peripheral Myelin Protein 22 (Pmp22). The extra copy of Pmp22 results in excessive PMP22 protein causing the neurophathy associated with CMT type 1A. Although there is no way to remove the extra copy of the gene, even subtle decreases in Pmp22 expression have shown promise against this inherited neuropathy in laboratory models.

In a recent paper, Inglese et al. 2014, describe an interesting new approach used to identify compounds that effectively decrease Pmp22 expression using a novel gene editing strategy and reporter-based screen. Their challenge was to create an assay that accurately represented endogenous Pmp22 expression including both transcriptional and post-transcriptional regulatory mechanisms, while maintaining the sensitivity required to detect subtle changes in expression in a loss of signal assay in a format compatible with microtiter 1536-well quantitative high-throughput screening (qHTS). Continue reading

Curiosity Unleashed: Wisconsin Science Festival 2014

Our buggy conversation starter at the Wisconsin Science Festival

Our buggy conversation starter at the Wisconsin Science Festival

“Oh wait Mom, look, bugs!”

And she was off. The next thing I knew she had a pale greenish-blue tobacco hornworm caterpillar in her hand.

“I’ve never seen anything like this before.” Nor had I.

She turned it upside down and started tickling its legs with a finger of her free hand.

“What does it eat? What will it turn into? How big will it get?” Rapid fire questions at the student who was manning the table of bugs. “Ooo cool. Look Mom he has a stick bug on his shirt. What does a woolly bear caterpillar become? What’s the name of that beetle? Where does it live? Why is that hornworm so much more active than the one I’m holding?”

We both took a really good look at the beating heart just underneath the dorsal skin of the very active hornworm that was about to pupate.

That was one adventure. There were many more.

Promega was one of many sponsors of this year’s Wisconsin Science Festival: Curiosity Unleashed 2014. Continue reading

Tips for a Social Media User

Social%20Networks_smallerAs an HR professional, I attend different seminars and conferences to obtain credits for my HR certification. We had a SHRM (Society for Human Resources Management) state conference last week, and I learned all kinds of new strategies involving recruitment, succession planning, employee engagement and change management. One topic was present in every session I was in: social media. How to recruit through social media, engage employees in social media, and how to maintain your company and personal brand through social media.

One session in particular was focused solely on social media, and how it affects the processes of hiring, discipline and discharge. Continue reading

Extraction of Plant DNA Made Easy

By Trillium1946 at en.wikipedia (Transferred from en.wikipedia) [Public domain], from Wikimedia Commons

My one attempt at working with plant DNA when I was at the lab bench was trying to create a shotgun library from a rice BAC. Never have I needed to isolate nucleic acid from the source material, but based on my conversations with plant scientists, it can be problematic endeavor between the tough tissue and the compounds that can copurify during extraction and inhibit downstream applications. And if you want to isolate DNA or DNA from plant samples in an automated format, that just adds to the difficulties. Here I review an Applications in Plant Sciences article that compares DNA isolation using the Maxwell® 16 System with two other methods on 25 different plant species samples. The authors note that Promega provided the Maxwell® 16 instrument, DNA isolation cartridges and advice on its use. Continue reading

Choosing Your Subcloning Strategy

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.

Use of Cell-Free Technology to Evaluate Nuclease (TALEN) Activity on Target DNA

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

Transcriptional activator-like effector nucleases (TALENs) have rapidly become a technique of choice for precision genome engineering. TALENs are custom-designed nucleases that consist of a modular DNA-binding domain fused to a monomeric, C-terminal FokI nuclease domain (1). TALENs work in pairs and are designed to recognize and bind to tandem-oriented sequences in genomic DNA, separated by a short spacer (15–30 bp). TALEN binding causes dimerization and activation of the FokI nuclease domains, which results in cleavage of the DNA within the spacer region. Small insertions or deletions (indels) are frequently introduced at this site, as the result of errors made during DNA repair by nonhomologous end-joining (NHEJ). These indels can be up to several hundred base pairs in length and result in frameshift mutations that lead to the production of truncated or nonfunctional proteins.

Successful use of TALENs for inducing targeted mutations has been reported in many conventional models, for example: mice, Xenopus and D. melanogaster. TALENs are also reported to be functional in a variety of other invertebrate arthropods, including mosquitos,silkworm and cricket. A recent publication (2) illustrates the use of TALEN technology for the genetic manipulation in P. dumerilii (marine ragworm). Continue reading

Highlights from ISHI25

ISHI25 logoLast week (September 29–October 2), I was one of almost 1,000 people who attended the 25th International Symposium on Human Identification (ISHI25) in Phoenix, Arizona. This scientific meeting brings together DNA analysts from forensic and paternity labs, research scientists and others with an interest in DNA-based identification to learn about new technologies, policy and process changes, and current and future trends in DNA typing. There were so many great presentations and learning opportunities, how do I pick just a few of them to highlight?

Continue reading

Free Webinar: Protein Reference Materials for Mass Spectrometry

MSextractcroppedOnce the domain of analytical chemistry labs, mass spectrometry instruments are now used in basic life science research, drug discovery research, environmental and industrial laboratories, and in many other biological laboratories. These instruments, which are used to perform critical analyses, need to be monitored for performance and quality.

How do you determine the system suitability and quality for your experiments? How do you evaluate a sample preparation protocol for mass spec analysis to ensure you get the best results possible without introducing artifacts? And, if you are trying to optimize the instrument method, what standard do you use ?

Currently, there is no commercially available reference reagent to help you monitor and test all LC (liquid chromatography) and MS (mass spec) parameters, particularly sensitivity and dynamic range, in a single run.

But what if there were an optimized peptide mixture that could report all key instrument performance parameters in a single run? Even better, what if that mixture came with free software to make analysis of all of the data you can generate even easier?

The upcoming free webinar: The 6 × 5 LC-MS/MS Peptide Reference Mixture and Software Analysis Tool describes such a standard reagent and analysis software.
If you are interested in learning about standardized reagent and software to help you monitor your instrument or optimize methods or protocols, register for this free webinar today!

Detecting Inhibition of Protein Interactions in vivo

Protein Interactions with NanoBRETIn a paper published in the September issue of ACS Medicinal Chemistry Letters, researchers from GlaxoSmithKline in the UK and Germany report on the discovery, binding mode and structure:activity relationship of a new, potent BRPF1 (bromodomain and PHD finger containing protein family) inhibitor. This paper came to our attention as it is one of the first publications to apply Promega NanoBRET technology in an vivo assay that reversibly measures the interaction of protein partners. The technology enabled the identification of a novel inhibitor compound that disrupts the chromatin binding of this relatively unstudied class of bromodomain proteins.

What exactly are bromodomains and why do they matter?
Bromodomains are regions (~100 amino acids) within chromatin regulator proteins that recognize and “read” acetylated lysine residues on histones. These acetylated lysines act as docking stations for regulatory protein complexes via binding of the bromodomain region. Because of their role in chromatin binding and gene regulation, bromodomains have attracted interest as potential targets for anti-cancer treatments. Although some bromodomain-containing proteins (e.g., those in the bromodomain and extraterminal domain (BET) subfamily) are well characterized and have been identified as potential therapeutic targets, others are less well understood. Continue reading