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Plasmids 101: Simplify cloning with in vivo assembly

Posted by Guest Blogger on Oct 18, 2018 8:37:05 AM

This post was contributed by Jake Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology.

Plasmid cloning is an essential part of any molecular biology project, yet very often, it is also a bottleneck in the experimental process. The majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination pathway that is present even in common lab cloning strains.

This intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together pieces of linear DNA through short homologous sequences at their termini, and likely functions as a bacterial DNA repair mechanism. The pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested so far.

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Topics: Plasmids 101, Plasmid Cloning

Golden Gate Assembly upgrades: More fragments, faster assembly, and higher fidelity

Posted by Guest Blogger on Oct 11, 2018 8:30:35 AM

This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs.

Golden gate assembly limitations

Embraced by the synthetic biology community, Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway engineering and optimization. However, current best practices for assemblies of more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such as enzyme efficiency, stability, and buffer compatibility have placed practical limits on single- or two-step assemblies.

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Topics: Plasmid Cloning

The time and cost required to make a plasmid

Posted by Jennifer Tsang on Sep 11, 2018 9:48:42 AM

Have you ever wondered how long it takes to make a plasmid? Or have you thought about how much total time you spend in the lab cloning before you can start on your experiment? What about all the reagents you need to order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally dive deep into experiments.

Since one million plasmids shared is upon us at Addgene, we started wondering, just how much time and money do researchers spend cloning? How much does material sharing speed science?

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Topics: Plasmid Cloning, Lab Tips

Plasmids 101: 5 factors to help you choose the right cloning method

Posted by Michael G. Lemieux on Aug 21, 2018 8:31:59 AM

You’ve spent days and weeks thinking of an amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. You’re going to engineer the best thing since CRISPR; you are ready to clone! But...how?

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Topics: Plasmids 101, Plasmid Cloning

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

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