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Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

Posted by Guest Blogger on Oct 27, 2015 11:00:00 AM

 This post was contributed by guest bloggers Erik L. Snapp and Lindsey M. Costantini.

"You underestimate the power of the Dark Side."

--Darth Vader in "Return of the Jedi"

While Vader was referring to the evil side of a mystical "Force," this quote is equally applicable to many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general.

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Topics: Imaging, Fluorescent Proteins

Tips for Screening with Yeast Two Hybrid Systems

Posted by Jessica Welch on Oct 22, 2015 10:30:00 AM

Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions.

The 1980s saw a flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers also discovered the modular format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual activities when separated (2), and that DBD and TAs from different systems could be combined effectively (3).

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Topics: Plasmid Technology, Yeast

The Future of Research Symposium Boston 2015

Posted by Guest Blogger on Oct 20, 2015 10:30:00 AM

The second Boston Symposium on the Future of Research will be held from 22-24 October. This blog has been contributed by guest blogger and Future of Research Symposium organizer, David T. Riglar PhD. Here, Dr. Riglar discusses one of the six panel discussions to be held at the Future of Research Symposium Boston 2015 – Academic Data and the Labor Market. For more information on the symposium as a whole and for registration please go to http://futureofresearch.org/boston/.

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Topics: Career, Career Readiness

Cpf1: A New Tool for CRISPR Genome Editing

Posted by Mary Gearing on Oct 14, 2015 10:30:00 AM

This post was updated on Dec 5, 2017.

In 2015, Zetsche et al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher on CRISPR/Cas9. 

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Topics: Genome Engineering, CRISPR

Tips from the Repository Trenches: Using Barcodes to Track Samples

Posted by Amanda Hazen on Oct 6, 2015 10:30:00 AM

Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received as DNA in a microcentrifuge tube. Once a sample enters Addgene’s lab, a series of events is triggered. Each step is tracked through barcoded tubes that are scanned into our Laboratory Information Management System (LIMS), a database we use to keep track of and manage all of the samples that come through our doors. It would be a nightmare if we didn’t have a simple way to track all of our 40,000 samples available for distribution!

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Topics: Inside Addgene, Lab Tips

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