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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

Modulate the Activity of 17 Signaling Pathways with One Kit!

Posted by Kendall Morgan on Mar 22, 2016 10:30:00 AM

When cancers are treated with drugs designed to hit them right where it hurts, the effects are often remarkable but fleeting.

“What’s been shown by others is that, in a relatively short amount of time, cancers become resistant to drugs, particularly targeted therapies,” said Kris Wood of Duke University Medical Center. “While we do not yet have a comprehensive understanding of how cancers become resistant, an emerging theme is that they do so by activating signaling pathways controlling properties like growth, survival, and differentiation.”

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Topics: Plasmid Technology, Cancer, Plasmid Kits

Minigenomes - a Safe Way to Study Dangerous Viruses

Posted by Guest Blogger on Mar 17, 2016 10:30:00 AM

This post was contributed by guest blogger Tessa Cressey.

The highly pathogenic Ebola virus belongs to the group of nonsegmented negative sense RNA viruses, along with other viruses that cause disease in humans such as measles, mumps, and rabies. Research on Ebola virus has been limited, in part, due to the necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard, minigenome systems, such as the one developed for Zaire ebolavirus (EBOV) are extremely useful, allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4).

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Topics: Viral Vectors

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