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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

Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

Posted by Brook Pyhtila on Mar 1, 2016 10:30:00 AM

Over the past decade, scientists have developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on. In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to put together DNA fragments.

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Topics: Plasmid Technology, Plasmids 101, Protocols, Plasmid Cloning

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

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