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Plasmids 101: Terminators and PolyA signals

Posted by Julian Taylor-Parker on Mar 31, 2016 10:30:00 AM

Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation, elongation, and termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination. Read on to learn more!

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Topics: Plasmid Elements, Plasmids 101, Plasmids

Plasmids 101: Cre-lox

Posted by A Max Juchheim on Jan 13, 2015 10:47:00 AM

In previous posts for our Plasmids 101 series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers (just to name a few). In this edition, we’re going to take a look at a very interesting tool that can be used for creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination.

What is Cre-lox?

The Cre-lox system is a technology that can be used to induce site-specific recombination events. The system consists of two components derived from the P1 bacteriophage: the Cre recombinase and a loxP recognition site. The P1 bacteriophage uses these components as part of its natural viral life-cycle, and researchers have adapted the components for use in genome manipulation.

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Topics: Plasmid Elements, Plasmids 101, Plasmids

Plasmids 101: Protein tags

Posted by Eric J. Perkins on Dec 11, 2014 11:26:00 AM

Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon, they have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection; however, those are just one (admittedly large) class of common fusion protein tags. Biochemists and molecular biologists who need to overexpress and purify proteins can face any number of technical challenges depending on their protein of interest. After several decades of trying to address these challenges, researchers have amassed a considerable molecular tool box of tags and fusion proteins to aid in the expression and purification of recombinant proteins.

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Topics: Plasmid Elements, Plasmids 101, Plasmid Tags

Choosing Your Perfect Empty Backbone

Posted by Lianna Swanson on Aug 19, 2014 11:39:33 AM

Vectors (or empty backbones) are frequently used in molecular biology to isolate, multiply, or express the insert they carry in the target cell. These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under various conditions. The first thing you'll need to decide when running your experiment, is which vector will best suit your needs?

At Addgene, we have a vast collection of empty backbones that have been designed, tested, and published by academic scientists. To help you find the vector that fits your experiments, I've described below some of the most frequently requested vectors in our repository and will discuss some of the features you may want to consider as you make your choice.

The first and most important thing you need to know is your expression system or environment. The host organism will determine the type of vector that you will need. You will also have to make sure that your plasmid has been incorporated into the host organism, usually achieved with the proper selection marker or antibiotic resistance.

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Topics: Plasmid Elements, Plasmids

Plasmids 101: The Promoter Region – Let's Go!

Posted by Kendall Morgan on Apr 3, 2014 4:05:00 PM

Thus far in our Plasmids 101 series we've worked our way through the plasmid map: antibiotic resistance, origin of replication, and so on. Up to this point we can replicate our plasmid and make sure cells maintain it; the next step is getting the plasmid to express our gene of interest. Enter the promoter-- the element responsible for initiating the transcription of your insert into RNA.

In practice, the term "promoter" describes the combination of the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed. 

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Topics: Plasmid Elements, Plasmids 101

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