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Arabidopsis in Education: How the Arabidopsis Biological Resource Center Brings Plant Science to Life

Posted by Guest Blogger on Apr 12, 2018 9:02:19 AM

This post was contributed by guest blogger Courtney Price, the Education & Outreach Specialist for the Arabidopsis Biological Resource Center and the Center for Applied Plant Sciences at The Ohio State University.

Established in 1991, the Arabidopsis Biological Resource Center (ABRC) is one of two global stock centers for Arabidopsis thaliana (Arabidopsis). Our mission is to collect, preserve, reproduce and distribute seeds, DNA and other resources for Arabidopsis and related species. Located at The Ohio State University in Columbus, Ohio, ABRC ships more than 100,000 samples to researchers and educators in 60 countries each year.

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Topics: Plant Biology, Education

When Fidelity Matters: A frank discussion about ligase fidelity

Posted by Guest Blogger on Mar 6, 2018 8:49:43 AM

This post was contributed by guest blogger Greg Lohman, a biochemistry researcher at New England Biolabs.

When do you need a high fidelity ligase—and when is an alternative ligase a better choice? And what is ligase fidelity anyway? Let’s talk about it.

DNA ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols, including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction, and circularization of dsDNA (6). Less commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq ligase is a NAD+-dependent DNA ligase from a thermostable bacterium that can survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C).

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Topics: Plasmid Cloning

CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

Posted by Guest Blogger on Feb 13, 2018 9:51:55 AM

This post was contributed by guest blogger Matthew J. Niederhuber, a graduate student at UNC Chapel Hill.

Chromatin immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where a protein binds genome-wide, and has been widely applied in many model organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations. The enzyme-based CUT&RUN method overcomes many of these limitations and makes it easier for you to map protein-DNA interaction with limited biological materials.

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Topics: Plasmid Technology, Techniques

Technologies Enabled by NanoLuc® Luciferase

Posted by Guest Blogger on Feb 8, 2018 7:17:02 AM

This post was contributed by Kyle Hooper at Promega.

Researchers have been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”. One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes it so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information. Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available from the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

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Topics: Fluorescent Proteins

In Vivo Biotinylation of Bacterial Fusion Proteins

Posted by Guest Blogger on Jan 25, 2018 9:09:35 AM

This post was contributed by guest blogger Jon Backstrom, a biochemist in the Vanderbilt Eye Institute and Tonia Rex's lab.

A common strategy to determine the binding kinetics of a purified protein involves immobilization on a solid support. This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on a reduced glutathione matrix. The advantage of a fusion protein is the efficient purification of an already immobilized target protein. The disadvantage is that the GST moiety, which forms dimers, may influence binding kinetics of the target ligand. Another important consideration is whether the affinity of an experimental protein-ligand interaction approaches that of GST-glutathione.

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Topics: Plasmid Technology, Hot Plasmids, Techniques

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