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 lifecycle, and researchers have adapted the components for use in genome manipulation.

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Topics: Plasmid Technology, Plasmid Elements, Plasmids 101

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

ICYMI: Addgene’s Plasmids 101 eBook

Posted by Marcy Patrick on Oct 2, 2014 1:25:00 PM

We'd like to thank all of the contributing Addgenies that made this eBook possible: Melina Fan, Matthew Ferenc, Larissa Haliw, A. Max Juchheim, Caroline LaManna, Margo Monroe, Kendall Morgan, Jason Niehaus, Marcy Patrick, Lianna Swanson, Julian Taylor-Parker

We'd also like to thank our guest contributor: Gal Haimovich of greenfluorescentblog.org for helping us explain why things glow!

Addgene's Plasmids 101 eBook is here: Enjoy more time developing clever experiments and less time researching basic plasmid features – download the Addgene Plasmids 101 eBook!

Our goal was to create a one-stop reference guide for plasmids. We’ve combined our Plasmids 101 blog posts from the last year with some additional resources to create one downloadable PDF you can save to your desktop for easy reference. Highlights include our guide to fluorescent proteins, information about promoters and ORIs, and tips for naming your plasmids.  

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Topics: Plasmid Elements, Lab Tips, Plasmids 101

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 How To, Plasmid Technology, Plasmid Elements

Plasmids 101: Viral Vector Elements

Posted by Marcy Patrick on Jul 17, 2014 3:09:00 PM

The use of viral vectors in research is beneficial for a number of reasons, including but not limited to: helping to get difficult-to-deliver DNA into mammalian cells, increasing the efficiency of gene transduction, allowing for control over which cells are infected through viral pseudotyping, and ease of vector cloning and modification. At the most basic level, viral vectors consist of a viral genome that has been adapted into a plasmid-based technology and modified for safety through the removal of many essential genes and the separation of the viral components. Read on for a brief description of the viruses used to make these vectors as well as a table defining the major elements found within the plasmids comprising the viral vector systems.

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Topics: Plasmid Elements, Plasmids 101, Viral Vectors

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