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Plasmids 101: Origin of Replication

Posted by Kendall Morgan on Nov 11, 2020 9:23:38 AM

Originally published Feb 6, 2014 and last updated Nov 10, 2020.

Now that we know all about antibiotic resistance genes, let’s consider another basic element of any plasmid: the origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all of its control elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive within cells.

The replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but they still rely on the host machinery to make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs are held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs can be separated more readily at lower temperatures and allows the replication machinery room to come in and get busy making copies.

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Topics: Plasmid Elements, Plasmids 101, Plasmids

Plasmids 101: Screening Strategies Used in Plasmid Cloning

Posted by Jennifer Tsang on Sep 15, 2020 9:15:00 AM

If you’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone that contains the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic resistance marker). In a previous blog post, we covered how to use positive and negative selection in plasmid cloning

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Topics: Plasmids 101, Plasmid Cloning, Plasmids

Plasmids 101: What is a plasmid?

Posted by Margo R. Monroe on Apr 2, 2020 2:17:39 PM

Originally published Jan. 14, 2014 and updated Apr. 2, 2020.

Any newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. A plasmid is a small circular piece of DNA found in bacterial cells, and someone new to plasmids may need some extra guidance to understand the specific components that make up a plasmid and why each is important.

Our “Plasmids 101” series designed to educate all levels of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you with an overview of general molecular biology knowledge and techniques, and empower you with a firm understanding of the fundamentals. Our mission is to curate a one-stop reference guide for plasmids, so that you can spend less time researching the basics and spend more time developing cleverly designed experiments and innovative solutions necessary for advancing the field.


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Topics: Plasmids 101, Plasmids

Plasmids 101: Plasmid Incompatibility

Posted by Leah Schwiesow on Mar 31, 2020 9:15:00 AM

Plasmid incompatibility is defined as the inability of different plasmids to be maintained in one bacterial cell. In this Plasmids 101 post, we’ll cover why this happens, how it might affect your work, and how understanding it can be used for good. 

First, why are plasmids incompatible? Plasmid incompatibility occurs when multiple plasmids within one cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part of the plasmid that contains the origin of replication and the replication control machinery (Need a refresher on the Origin of Replication? See our Plasmids 101: Origin of Replication blog). 

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Topics: Plasmids 101, 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

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