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Plasmids 101: Mammalian Vectors

Posted by Marcy Patrick on Mar 25, 2014 11:15:00 AM

Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for example, and a toxin that kills bacteria may not have any discernable effect on mammalian cells. In this blog post we will discuss how mammalian plasmids differ from their bacterial counterparts, including how replication occurs and whether selection is necessary for transfected cells.

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Topics: Plasmid How To, Plasmid Technology, Plasmids 101

Plasmids 101: Yeast Vectors

Posted by Marcy Patrick on Feb 25, 2014 2:11:00 PM

In our first few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenence within an E. coli cell, but vectors can be widely utilized across many different cell types and each one requires different elements for vector propogation. This post, along with a future companion post on mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and explain how they differ from the bacterial elements previously described.

Why Do Scientists Use Yeast Vectors?

Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still share many of the same technical advantages that come with working with prokaryotes. This includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and a highly versatile DNA transformation system.

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Plasmids 101

Plasmids 101: How to Name Your Plasmid in 3 Easy Steps

Posted by Matthew Ferenc on Feb 13, 2014 8:00:00 AM

There are no universal rules for naming plasmids but here are some good guidelines to follow in order to ensure that people can quickly and easily identify what your plasmid contains and other important information.

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Topics: Plasmid How To, Lab Tips, Plasmids 101

Plasmids 101: Origin of Replication

Posted by Kendall Morgan on Feb 6, 2014 10:25:00 AM

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 melt more readily at lower temperatures. When DNA melts, it gives the replication machinery room to come in and get busy making copies.

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Topics: Plasmid How To, Plasmid Elements, Plasmids 101

Plasmids 101: Antibiotic Resistance Genes

Posted by Marcy Patrick on Jan 30, 2014 10:29:00 AM


Resistance to antibiotics is a widely used tool in molecular biology, yet scientists rarely stop to think about how much easier it makes our lives. Plasmid transformation into E. coli is a fairly inefficient process– just 1 out of 10,000 cells on average! Without some means of quickly determining which cells successfully received the correct plasmid, scientists would spend hours to days trying find their correct clones. Additionally, the presence of a plasmid is disadventageous from the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal DNA, costing additional resources to maintain the plasmid. Adding an antibiotic resistance gene to the plasmid solves both problems at once – it allows a scientist to easily detect plasmid-containing bacteria when the cells are grown on selective media, and provides those bacteria with a pressure to keep your plasmid. Viva la (bacterial) resistance! 

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Plasmids 101

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