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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

Illuminating Epigenetics with A FRET Based Biosensor

Posted by Emma Markham on Nov 19, 2015 10:30:00 AM

Epigenetics has recently been hitting the headlines, with sotires like the potential devastation of the palm oil industry through epigenetic effects on the Cover of Nature. So what is epigenetics and what tools are available to study it?

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Topics: Hot Plasmids, Fluorescent Proteins, Techniques

The Materials Science of Optogenetics Experiments

Posted by Guest Blogger on Sep 17, 2015 10:30:00 AM

This post is part of our Primer on Optogenetics and was contributed by guest blogger Derek Simon.

The surgeries and standard molecular neuroscience validation experiments we discussed last week are only half of the battle when using optogentics to answer a research question. The flip side of the optogenetics coin is materials science-based. Light is delivered to your opsin through a small piece of fiber optic cable implanted into the animal’s skull (right). The fiber optic cable is threaded throughand fixed to—an optical insulator called a ferrule (below). The fiber optic cable/ferrule is inserted into the target brain region using stereotaxic surgery and cemented to the animal’s skull using dental cement (a similar procedure as implanting a guide cannula). A fiber optic patch cable is then connected from laser to ferrule to deliver light pulses to the target brain region.

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Topics: Optogenetics, Techniques, Primer on Optogenetics

Plasmids 101: Golden Gate Cloning

Posted by Mary Gearing on Aug 27, 2015 10:30:00 AM

Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful within both the synthetic biology and genome engineering fields. We’ll walk you through how to apply this precise and easy-to-use system to your cloning efforts.

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Topics: Plasmid How To, Synthetic Biology, Plasmids 101, Protocols, Techniques, Plasmid Cloning

Another Pathway into Cells: iTOP

Posted by Mary Gearing on Jun 23, 2015 4:37:00 PM

Primary cells recapitulate the natural biology of a cell type of interest better than immortalized lines derived from the same cell type; however, their usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen’s lab suggests that primary cells may be better transduced using only protein. Read on for a description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.

 

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Topics: CRISPR, Techniques

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