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When Fidelity Matters: A frank discussion about ligase fidelity

Posted by Guest Blogger on Mar 6, 2018 8:49:43 AM

This post was contributed by guest blogger Greg Lohman, a biochemistry researcher at New England Biolabs.

When do you need a high fidelity ligase—and when is an alternative ligase a better choice? And what is ligase fidelity anyway? Let’s talk about it.

DNA ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols, including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction, and circularization of dsDNA (6). Less commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq ligase is a NAD+-dependent DNA ligase from a thermostable bacterium that can survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C).

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Topics: Plasmid Cloning, Plasmids

Cloning Mammalian Cells with the Agarose Method

Posted by Guest Blogger on Sep 7, 2017 8:17:41 AM

This post was contributed by guest blogger Iris Lindberg, Professor at the University of Maryland School of Medicine.

In the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings to isolate colonies of antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning rings, the remainder of the colonies on the plate dried out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since only wells with one cell can give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require extra serum to survive at low densities - adding to the expense of dilution cloning.

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Topics: Plasmid Cloning, Molecular Biology Protocols and Tips, Plasmids

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: Plasmid Cloning, Plasmids

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

Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

Posted by Beth Kenkel on Dec 15, 2016 10:57:02 AM

Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an alternative method that can be used for cloning or genome engineering and is based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from the lambda red bacteriophage and its use as a genetic engineering tool is frequently called recombineering - short for homologous recombination-mediated genetic engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has the flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA. 

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Topics: Plasmid Cloning, Plasmids

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