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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

Plasmids 101: Common Lab E. coli Strains

Posted by Matthew Ferenc on Nov 7, 2014 9:56:00 AM

This post was updated on Nov 14, 2017.

You've worked hard designing your plasmid – you carefully selected the optimal promoter for your gene of interest, painstakingly cloned into the perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent protein downstream, separated by an IRES element. You did a lot of work! But let’s take a moment to recognize your little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria.

It’s hard to count the number of different commercial strains of E. coli currently available  a quick Google search suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression. If you were to include customized strains, the number is probably in the thousands! The goal of this article is to provide enough background for you to distinguish the features of any common lab strain and determine whether it is appropriate for propogating your plasmid or carrying out your experiment.

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Topics: Lab Tips, 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

Plasmids 101: Multicistronic Vectors

Posted by Melina Fan on Sep 9, 2014 4:20:00 PM

Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters before so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work, and how to get started with them.

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Topics: Plasmid Technology, Plasmids 101

Plasmids 101: How to Verify Your Plasmid

Posted by Lianna Swanson on Aug 28, 2014 11:34:00 AM

Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into your functional experiments! Whether you’ve cloned the plasmid yourself or obtained it from a colleague down the hall, it is always a good idea to take some time to confirm that you are working with the correct construct, and verify that the plasmid you received matches the expected sequence. Here at Addgene, we use NGS-based quality control to confirm the sequence of all the plasmids we distribute. This method is time-intensive, so we recommend two other methods for quick plasmid verification: Sanger sequencing and diagnostic restriction digest.

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Topics: Plasmid How To, Lab Tips, Plasmids 101

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