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Choosing the B(right)est Fluorescent Protein: Photostability

Posted by Guest Blogger on Jun 8, 2017 10:30:00 AM

This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced microscopy, University of Amsterdam.

The previous post in this series described a practical approach to selecting a bright fluorescent protein. In the second post of this series, we will discuss how to select a photostable fluorescent protein.

Photobleaching is the irreversible destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017) and photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent proteis should emit a stable fluorescence signal, showing no or little deterioration or change of the signal during the course of the experiment.

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Topics: Fluorescent Proteins, Choosing the Brightest Fluorescent Protein

A Practical Approach to Choosing the B(right)est Fluorescent Protein

Posted by Guest Blogger on Jun 1, 2017 10:30:00 AM

This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam.

Before you decide which car you want to buy, it is worthwhile to test-drive a couple of candidates. Before you buy a new microscope, it is smart to look at (and through) a couple of models. Before you start a new project with fluorescent proteins, the best advice is to try a couple of promising variants to check how they perform under your experimental conditions. This is time well spent and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts explains how to critically assess the reported properties of fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein for your application.

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Topics: Fluorescent Proteins, Choosing the Brightest Fluorescent Protein

Enabling Precision Functional Genomics with the Target Accelerator Plasmid Collection

Posted by Guest Blogger on May 11, 2017 10:30:00 AM

This post was contributed  by Jesse S. Boehm, the Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT.

The notion of cancer precision medicine seems so simple! Take a patient’s tumor sample, use cutting edge genomic technologies to map the mutations that are present, and use prior knowledge (data connecting each genotype with vulnerabilities) to design a therapeutic strategy that works.

But, those darn cancers have revealed many tricks up their sleeves and most patients still don’t benefit from this approach. One central bottleneck is that most recurrently mutated cancer genes are rare and most of the individual variants found in tumors are exceedingly rare. As a result, how most of these “variants of unknown significance” (sometimes called “VUS”) function is unknown. How can we make a decision for each patient if the majority of information on each cancer clinical sequencing report includes rare variants that haven’t been characterized?

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Topics: Cancer

How to Design Your gRNA for CRISPR Genome Editing

Posted by Guest Blogger on May 3, 2017 11:00:00 AM

This Post was updated on May 3, 2017 with additional information and resources. 

This post was contributed by guest blogger, Addgene Advisory Board member, and Associate Director of the Genetic Perturbation Platform at the Broad Institute, John Doench.

CRISPR technology has made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post will discuss differences between these approaches, as well as provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's Validated gRNA Sequence Datatable.

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Topics: Plasmid How To, Genome Engineering, Lab Tips, CRISPR

Tips for Getting a Faculty Position

Posted by Guest Blogger on May 2, 2017 10:30:00 AM

This post was contributed by guest blogger Erik Snapp, Director of Graduate and Postdoctoral Programs at Janelia Research Campus.

Eight years ago, I decided to write a "how to" manual on applying for faculty positions in biomedical science. My motivation was to share my experiences from my own job search and my time on faculty search committees. Having successfully navigated the trials and tribulations of the process, I’ve provided guidance and mentoring to several people that found my insights helpful. All went on to get faculty positions at top state colleges, private universities, and medical schools.

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Topics: Career

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