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Plasmid Preps: Different Purity, Different Quantities, Different Uses

Posted by Paolo Colombi on Jun 16, 2020 9:25:46 AM

Not all plasmid preps are the same. Before purifying a plasmid from a bacterial culture, it is important to consider your experiment.  It will dictate the amount of DNA you need, and at which level of purity. Based on these premises we can classify a plasmid preparation in 3 different ways:

  • Transformation grade DNA
  • Cloning grade DNA
  • Transfection grade DNA
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Topics: Plasmid Cloning, Plasmids

Early Career Researcher Toolbox: Free Online Molecular Biology Tools

Posted by Beth Kenkel on Apr 14, 2020 9:15:00 AM

Primer design. Plasmid mapping. DNA sequence analysis. We all have our favorite tools for tackling these particular tasks, but they tend to be scattered about the internet. To help you keep your virtual molecular biology toolbox organized, today’s post features a list of free online molecular biology tools all in one place.

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Topics: Plasmid Cloning, Other Plasmid Tools, Plasmids, Early Career Researcher

Troubleshooting Your Plasmid Cloning Experiment

Posted by Guest Blogger on Sep 24, 2019 8:55:00 AM

This post was contributed by Oskar Laur, head of the custom cloning core at Emory University, and Paolo Colombi, a product development scientist at Addgene.

Cloning can be quite an arduous process. The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies screened might not contain the correct plasmid. There’s a lot that can go wrong! With all the steps in the cloning process, there are also many ways to troubleshoot the cloning experiment. Here are some tips that will help you with your cloning project, and hopefully obtain your coveted plasmid with no substantial delays.

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Topics: Plasmid Cloning, Plasmids

Plasmids 101: Positive and Negative Selection for Plasmid Cloning

Posted by Jennifer Tsang on Aug 22, 2019 8:43:39 AM

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.

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Topics: Plasmids 101, Plasmid Cloning, Plasmids

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: Plasmid Cloning, Molecular Biology Protocols and Tips, Plasmids

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