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Using Phosphoserine to Study Protein Phosphorylation

Posted by Guest Blogger on Jun 23, 2016 10:30:00 AM

This post was contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research in Madison, Wisconsin.

It is commonly cited that approximately one-third of cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array of model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of any of these phosphorylation events on the activity of a given protein?

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Topics: Plasmid How To, Synthetic Biology, Lab Tips, Techniques

Plasmids 101: Colony PCR

Posted by Guest Blogger on May 12, 2016 10:30:00 AM

This post was contributed by guest blogger Beth Kenkel, a Research Assistant in the Department of Pediatrics at the University of Iowa. If you're interested in guest blogging, let us know!

Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze product size. This blog post discusses some of the key things to consider when performing colony PCR.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

Addgene's Tips for Plasmid Quality Control

Posted by Julian Taylor-Parker on Jan 14, 2016 10:30:00 AM

One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through our quality control processes. Every plasmid we receive is rigorously verified before becoming available to the community.

This is no small task, however, at a repository that consistently receives around 200 new DNA samples every week. Here we will provide an inside look at the steps we take to verify the identity and quality of the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids.

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Topics: Plasmid How To, Inside Addgene, Lab Tips

Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

Posted by Mary Gearing on Dec 17, 2015 10:30:00 AM

If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step! Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

Plasmids 101: Golden Gate Cloning

Posted by Mary Gearing on Aug 27, 2015 10:30:00 AM

Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful within both the synthetic biology and genome engineering fields. We’ll walk you through how to apply this precise and easy-to-use system to your cloning efforts.

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Topics: Plasmid How To, Synthetic Biology, Plasmids 101, Protocols, Techniques, Plasmid Cloning

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