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CRISPR 101: Cas9 Nickase Design and Homology Directed Repair

Posted by Mary Gearing on Mar 15, 2018 8:59:40 AM

By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent gRNAs, can lower the probability of off-target editing. But that’s not all! New research from IDT (Integrated DNA Technologies) has shown that a nickase approach can improve homology directed repair (HDR) rates, provided you follow some simple design rules described below.

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Topics: CRISPR, CRISPR 101

Imaging Tools and Following a Passion for Basic Biology: Interview with Joachim Goεdhart

Posted by Tyler Ford on Mar 9, 2018 9:00:00 AM

Today on the Addgene Podcast, we have another interview conducted by European Outreach Scientist Benoit Giquel. Ben recently spoke with Joachim Goεdhart, a professor at the University of Amsterdam. In addition to creating and sharing many great fluorescent protein tools, Professor Goedhart is very active on Twitter and has helped us update many of our educational resources. Listen to hear all about Professor Goedhart, his lab, and some of the tools he’s developed.


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Topics: Fluorescent Proteins, Podcast

New Neuroscience Tool: The SF-iGluSnFr Glutamate Sensor

Posted by Tyler Ford on Mar 8, 2018 9:00:00 AM

In a previous blog post we discussed how fluorescent proteins can be used to construct biosensors, biological tools that monitor processes or detect molecules. Here we’ll be diving into the details surrounding SF-iGluSnFr, a recently upgraded biosensor designed to detect glutamate.

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Topics: Hot Plasmids, Fluorescent Proteins

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

The Importance of High Titer for AAV Transductions

Posted by Luke Hanley on Mar 1, 2018 9:11:54 AM

Many of us take comfort in the fact that it’s often not quantity, but quality that really matters. Well, it turns out this isn’t the case for using AAV. When it comes to infecting cells, titer, the amount of virus used, really does matter. (*psst*, quality definitely also matters).

Check out this post for a refresher on AAV titers

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Topics: Viral Vectors

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