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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

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: Stringent Regulation of Replication

Posted by Jason Niehaus on Dec 3, 2015 10:30:00 AM

Plasmids utilize their host cell's replication machinery in order to replicate. As described in our previous Origin of Replication post, DNA replication is initiated at the ORI and may be synchronized with the replication of the host cell's chromosomal DNA or may be independent of the host's cell cycle. 

Plasmids are said to be under stringent control of replication when they are dependent on the presence of initiation proteins synthesized by the host cell in order to start their own replication. In general, these types of plasmids tend to be low copy number. Conversely, plasmids that can initiate DNA replication independently of the host's initiation proteins are said to be under relaxed control, as they only require the host's replication machinery for elongation and termination. These types of plasmids tend to be high copy number.

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Topics: Plasmid Elements, Plasmids 101

Plasmids 101: A Brief History of Plasmids and an Improved eBook!

Posted by Marcy Patrick on Oct 29, 2015 10:30:00 AM

Bioblasts? Plasmagenes? In the 1940s and 50s, scientists were working to understand genetic cytoplasmic factors that could be transferred between cells. At the time, these extranuclear agents of heredity were thought of as everything from parasites, to symbionts, to genes and the labels applied to them were vague or contradictory, owing in part to the fact that very little was known about the role these factors played within an organism. 

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Topics: Plasmids 101, 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

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