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Plasmids 101: Restriction Cloning

Posted by Tyler Ford on Feb 18, 2016 10:42:06 AM

When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of how to do perform restriction digests.

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Topics: Plasmids 101, Protocols, Plasmid Cloning

REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

Posted by Mary Gearing on Feb 10, 2016 10:30:00 AM

Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular biology and genetics. It can be used to characterize the function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating mutations. In biomedical research, modeling patient mutations using SDM can help determine if a variant is causal for a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While commercial kits are available for making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized Plasmids After PCR).

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Topics: Protocols, Techniques, Plasmid Cloning

How to Lead a Great Meeting

Posted by Carissa Fish on Feb 9, 2016 10:30:00 AM

Meetings often get a bad rap as annoying interruptions to our “real” work. However, a well-run meeting can have quite the opposite effect. A great meeting should produce collaboration - a sense of dialogue and community among participants, clarification - new and useful information, and invigoration - a renewed energy for continuing the project after leaving the meeting. Follow the tips below to learn how you can run a top-notch meeting.

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Topics: Career, Inside Addgene, Career Readiness

Anatomy of a Plasmid Page at Addgene

Posted by Jessica Welch on Feb 4, 2016 10:30:00 AM

Have you ever found yourself bamboozled by all of the different kinds of information on our plasmid pages? Well, to help make the most of these pages, we've written this post to guide you through them and make the best use of all the information provided by your colleagues.

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Topics: Plasmid Elements, Inside Addgene

Components of CRISPR/Cas9

Posted by Joel McDade on Feb 2, 2016 12:00:00 PM

At their most basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post, we hope you will be caught up on much of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements of these different systems can vary (for example, read our posts on Cpf1 and eSpCas9/SpCas9-HF1 for some of the interesting properties of these exciting new nuclease tools).

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Topics: CRISPR 101

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