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You’ve worked hard to purify your gene of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells. Unfortunately, not every cell successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene of interest, but others will uptake plasmid backbones that re-ligated back on themselves.

Therefore, your cloning strategy needs to identify cells containing the plasmid construct you’re seeking. Fortunately, there are many ways to do this involving positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection in this blog post, but don’t worry, we’ll cover screens in a future post.

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.

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.

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?

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?