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Pushing the Limits of DNA Assembly

Posted by Guest Blogger on Sep 6, 2017 9:01:33 AM

This post was contributed by guest blogger Lydia Morrison from New England Biolabs.

What is DNA assembly? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA assembly available, including: Gibson Assembly®, BioBrick® Assembly, Golden Gate Assembly, and NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using homologous regions to guide the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of Type IIS restriction enzymes results in scarless assembly. Finally, the NEBuilder® HiFi DNA Assembly method from New England Biolabs® has minimal upfront requirements and allows you to expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments, and generate constructs for producing single-guide RNAs – but it also allows you to skip purification steps and end-repair steps with well-designed fragment overlap sections, while still creating scarless plasmid inserts. 

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Topics: Techniques, Plasmid Cloning

MXS Chaining

Posted by Leila Haery on Feb 7, 2017 10:30:00 AM

High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate each element into the final construct. This strategy can be used to build vectors with diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine the functions of different genetic elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette of fluorescent proteins and the availability of powerful imaging technologies, the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect structure and protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and Neveu, 2015).

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Topics: Fluorescent Proteins, Plasmid Cloning

Plasmids 101: Gateway Cloning

Posted by Maria Soriano on Jan 12, 2017 10:30:00 AM

When facing a cloning project, scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that will work well with a given set of resources, time, and experimental needs. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Keep reading to learn more about the Gateway cloning method and its advantages.

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Topics: Plasmids 101, Plasmid Cloning

Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

Posted by Michael G. Lemieux on Dec 8, 2016 10:30:00 AM

If you’re into cloning, you’re probably aware that there are several methodologies currently available for approaching it. These include the traditional restriction enzyme/ligase-mediated method, the more recently developed Gibson Assembly Cloning and Gateway® cloning technologies, as well as several others. Each method is unique and relies on specific components that are key to the cloning reaction. Understanding the specific components is essential for choosing the correct cloning method for your own experiments, and here we will focus on a unique gene that makes the popular GatewayTM method possible: ccdB. But what is ccdB, what role does it play in modern cloning, and why should you learn more about it? Read on to find out how ccdB can make your cloning experiments a little easier.

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Topics: Plasmids 101, Plasmid Cloning

FastCloning: A Newer, Simpler Cloning Technique

Posted by Guest Blogger on Nov 1, 2016 10:30:00 AM

This post was contributed by guest blogger Meghan Monroy, a graduate student in Protein Science at the University of Connecticut.

Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques are some of the most widely used techniques in the biological sciences and constitute foundational elements of biomedical research. Traditional restriction cloning is one of the oldest of these techniques and is a multi-step process consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely performed by many labs, a variety of other cloning techniques with higher efficiency and simpler procedures have been developed. Some of these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning, and overlap extension PCR. Although each type of cloning has its advantages, most scientists still encounter several struggles with these techniques: unwanted mutations due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive time requirements. FastCloning is a simpler yet reliable cloning technique that was developed by Chaokun, et al., in 2011. This method is ligation independent, it does not require purification of insert or vector products, nor does it require the use of specific sequences. Read on to learn how easy this process is and to get tips for applying it in your own lab.

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Topics: Plasmid Cloning

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