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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

9 tips for a successful postdoctoral experience

Posted by Guest Blogger on Oct 9, 2018 8:21:40 AM

Today's postdoctoral fellows (PFs) face a number of challenges ranging from long periods of training to limited job opportunities in academia - the main reason most people enter postdoctoral training. Similarly, there are several factors to consider when selecting a postdoc mentor and lab. These topics have been addressed in numerous essays and workshops (see the Careers essays in the journals Nature and Science, for example).

This blog post is about how to get the most out of your postdoctoral training experience. If you're going to commit to doing a postdoctoral fellowship, what are you signing up for and how can you do it well? Below, I briefly describe some tips for a successful postdoc fellowship.

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Topics: Career Readiness, Career

New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

Posted by Alyssa Cecchetelli on Oct 4, 2018 8:44:18 AM

CRISPR pooled libraries have allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate gene function. CRISPR libraries can be used to knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives.

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Topics: CRISPR, pooled libraries

Is this the right place for me? 8 tactics for choosing a lab

Posted by Joanne Kamens on Oct 2, 2018 8:56:53 AM

Why is choosing the right lab such a big deal? It’s actually something you CAN choose and it will make a huge difference for your future career and life. You might see a lab head as choosing you, but in reality, you are giving your hard work and talent for many years (at a very low salary I might add). You have a right and responsibility to choose a lab where you can thrive and do your best work. This post is focused on choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes me when the most analytic scientists seem to toss data-driven reasoning out the window when making decisions. We think scientists make choices based on logic and reason, but often our decisions are based on emotions and assumptions. I’m not just talking about simple things like “Which route should I take to get to work?”; we often make important life decisions without much logic - even choosing a training lab where we will spend the next 6 years.

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Topics: Career, Career Readiness

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