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Getting the Most from Your Lentiviral Transduction

Posted by Meghan Rego on Apr 7, 2016 10:30:00 AM

Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express a gene of interest. While the general approach for using lentivirus, infect and select, seems simple, in actuality, many find using lentivirus to be time consuming, difficult, and lacking in reproducibility. Read on for some tips for getting the most out of your lentiviral transduction experiments.

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Topics: Lab Tips, Viral Vectors

Providing Plasmids to Researchers in Developing Countries: Addgene and Seeding Labs Team Up

Posted by Tyler Ford on Apr 5, 2016 10:30:00 AM

Researchers in developing countries often find themselves on the front lines of emerging global challenges. However, these same researchers face significant barriers to scientific discovery and career development. They are working with limited resources but limitless potential. We see it as part of our mission to enable all researchers and are proud to announce that we are teaming up with Seeding Labs to better serve those in developing countries.

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Topics: Scientific Sharing, Inside Addgene

Plasmids 101: Terminators and PolyA signals

Posted by Julian Taylor-Parker on Mar 31, 2016 10:30:00 AM

Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation, elongation, and termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination. Read on to learn more!

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Topics: Plasmid Elements, Plasmids 101

Plasmid Cloning by PCR

Posted by Various Addgenies on Mar 29, 2016 10:30:00 AM

In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest. You can use similar processes to add overhangs to your insert of interest for Gibson assembly. The steps following primer design and the PCR process itself are very similar to those outlined in our restriction cloning post with a few quirks specific to the PCR cloning process - please check out that post if you need a more detailed refresher on the downstream steps.

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Topics: Protocols, Plasmid Cloning

Optimizing Donor DNA for Enhanced CRISPR Genome Editing

Posted by Guest Blogger on Mar 24, 2016 10:30:00 AM

This post was contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab.

CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently than NHEJ and the efficiency of these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision.

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Topics: Genome Engineering, CRISPR

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