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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

Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

Posted by Mary Gearing on Dec 17, 2015 10:30:00 AM

If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step! Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

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

CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

Posted by Guest Blogger on Feb 18, 2015 10:09:22 AM

The following post was contributed by Daniel Bauer and Matthew Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations behind this research.

 

Using CRISPR/Cas9 for Targeted Genomic Deletions

We were inspired to produce intrachromosomal deletions based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One observation was an inverse relationship between deletion size and frequency [3].

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Topics: Genome Engineering, Lab Tips, CRISPR, Protocols

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