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CRISPR 101: Non-Homologous End Joining

Posted by Guest Blogger on Apr 16, 2015 11:45:08 AM

This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill.

One advantage to using the CRISPR/Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses. After the initial cut, the next steps in the process involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways  Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could be relevant when planning your experiment. This blog has previously considered the HDR pathway; below we’ll discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome.

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Topics: Genome Engineering, CRISPR, CRISPR 101

Plasmids 101: Control Plasmids

Posted by Chari Cortez on Apr 9, 2015 11:29:00 AM

There are many, many different types of experiments carried out by scientists every day. Although the designs and outcomes may vary, one thing should be present in every experiment-based investigation of a hypothesis: proper controls!

For every experiment, an investigator needs a standard against which the results can be compared; results from an experiment lacking the proper controls are invariably inconclusive and unreliable. Proper controls provide the constant variables that enable the correct interpretation of the effect of the independent variable you are testing. Importantly, they demonstrate the functionality of your experimental system and help identify opportunities for troubleshooting or optimization within your experiment. Read on to learn more about the various controls that can be used for plasmid-based experiments.

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Topics: Lab Tips, Plasmids 101

Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

Posted by Guest Blogger on Apr 7, 2015 12:21:00 PM

This post was contributed by Adam Chin-Fatt, a Ph.D. student at the University of Western Ontario. Adam summarizes Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize the concepts discussed in the paper.

The transcriptional control of multiple loci is deftly coordinated by the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these cellular behaviors often fall short with the generation of various flux imbalances. The conventional approach has typically been to either systematically delete/overexpress endogenous genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However, these approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have been limited to the overexpression of various metabolites in Escherichia coli or Saccharomyces cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes.

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Topics: Plasmid Technology, Genome Engineering, Synthetic Biology, CRISPR

Management for Scientists: Managing vs. Leading

Posted by Joanne Kamens on Mar 31, 2015 12:08:00 PM

This is the fifth in a 5 part series on Management for scientists. Subscribe to the Addgene Career Advice Posts here.

We are inundated with articles and books on the topic of leadership. Perhaps one of your advisors or mentors has urged you to work on developing your “leadership skills”. Leadership is prized at all levels of an organization and is also one of the most common criteria required for a promotion. Yet little explanation is given for how someone can or should demonstrate this quality.  

I am often asked to give career seminars on Leadership Skills. After attempting to put together such a presentation many times, I could never actually figure out what skills were really leader-specific. How is leadership different from good management? Aren't all career skills leaderhsip skills when done well? Do you just have to know it when you see it?

Check out Joanne's Reddit AMA

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Topics: Career, Management for Scientists

500,000 Plasmids Shipped and Counting!

Posted by Caroline LaManna on Mar 26, 2015 11:24:00 AM

Yesterday we hit a huge milestone here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around the world in ~10 years!

We're extremely proud to be able to support scientists by bringing you all the plasmids you need. We're focused on our mission – documenting your plasmids, storing them, providing quality control and customer support, making plasmids easy for you to find and request in our online collection, and shipping them to you quickly.

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Topics: Fun, Scientific Sharing, Inside Addgene

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