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Your Lentiviral Plasmid FAQs Answered

Posted by Kendall Morgan on Apr 23, 2014 9:08:00 AM

Lentiviruses are useful and efficient tools to introduce your gene of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can infect dividing and non-dividing cells. 

Addgene has an extensive collection of lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown, Tet and Cre-regulated expression, CRISPR genome editing, and more. Not surprisingly, we receive many questions from scientists all over the world looking for some additional information or clarification on these vectors. Read on to find the answers to our most frequently asked lentiviral questions.

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Viral Vectors

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

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