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5 Tips for Troubleshooting Viral Transductions

Posted by Leila Haery on Aug 11, 2016 10:23:59 AM

An estimated 320,000 viruses can infect mammals. Even more abundant are the Earth’s estimated 1031 bacteriophages (viruses that infect bacteria), many of which are doing important work in our microbiomes. Given that viruses are everywhere and doing everything, it can be annoying when we try to use them in an experiment and they don't do anything.

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Topics: Viral Vectors

CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

Posted by Guest Blogger on Aug 9, 2016 10:30:00 AM

This post was contributed by guest blogger, Aneesh Karve, CTO at Qult Data. This post was originally published on the Quilt Genomics Blog and is republished here with permission.

Quilt is a collaborative database for genomics. In this article, Quilt CTO Aneesh Karve, shows how to design experiments that work anywhere in the genome. Aneesh's research interests include proteomics, machine learning, and visualization for big biology.
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Topics: CRISPR

Editor's Choice, July 2016

Posted by Tyler Ford on Aug 5, 2016 11:00:00 AM

To better highlight the great content contributed by our bloggers each and every month, we've decided to start an "Editor's Choice" series. Each month, I'll summarize the most popular post of the month and point out one or more additional posts that deserve a peek in case you missed them.

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Topics: CRISPR, Fluorescent Proteins, Editor's Choice

Google Forums Round Up: First Impressions of NgAgo

Posted by Guest Blogger on Aug 4, 2016 10:30:00 AM

Update (November 18, 2016): Researchers from a variety of institutions recently reported their inability to recapitulate the results of Gao et al 2016 in a letter to Protein & Cell.

Update (August 3rd, 2017) THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITING BY THIS TOOL

This post was contributed by guest blogger Pooran Dewari. Any views in this post are those of the guest blogger and do not necessarily represent the views of Addgene. Addgene performs Sanger sequencing on select regions of all distributed plasmids as part of quality control, but does not perform functional tests.

The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is faring with users in a minute, but, to start, let's review why NgAgo is in the spotlight and take a moment to remember that NgAgo has only been available for genome editing for a few months. More time is required for its optimization and development before it can truly be pitted against CRISPR head-to-head. 

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Topics: Genome Engineering, CRISPR

Site Directed Mutagenesis by PCR

Posted by Guest Blogger on Aug 2, 2016 10:30:00 AM

This post was contributed by guest blogger, Kristian Laursen from Cornell University.

Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.

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Topics: Lab Tips, Protocols

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