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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

3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

Posted by Guest Blogger on Sep 5, 2017 9:58:42 AM

This post was contributed by guest bloggers Dominik Paquet and Dylan Kwart from Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in NYC.

The CRISPR/Cas9 system is a versatile tool for precise gene editing in many organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred to as ‘indels’. While this is convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence changes.

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Topics: CRISPR

Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies

Posted by Guest Blogger on Jul 28, 2017 9:30:50 AM

This post was contributed by guest blogger Jon Chow, an immunology PhD student at Harvard University.

In my previous two posts, I’ve described the fundamentals of how to work with Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study gene function. In this final installment, I’ll provide a brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG). 

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Topics: Drosophila, Quick Guide to Drosophila

Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

Posted by Guest Blogger on Jul 21, 2017 8:48:55 AM

This post was contributed by guest blogger Jon Chow, an immunology PhD student at Harvard University.

In this second post in our quick guide to working with Drosophila, you’ll learn how to maniupate expression of your favorite gene (YFG) in flies. Read the first post here.

Once you’ve identified some fly stocks and other reagents of interest, the next question to ask is what to do with them. In some cases, there might be a mutation that disrupts the function of YFG. You could compare this mutant fly to one lacking the mutation in the same genetic background. In other cases, YFG or one of its mutant variants will need to be overexpressed or knocked down. To do this, Drosophila geneticists use the Gal4/UAS system. This incredibly useful, yet simple system allows you to systematically study gene function with temporal control and cell-type specificity!

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Topics: Lab Tips, Drosophila, Quick Guide to Drosophila

Quick Guide to Working with Drosophila Part 1: Getting Started with Flies

Posted by Guest Blogger on Jul 13, 2017 10:30:00 AM

This post was contributed by guest blogger Jon Chow, an immunology PhD student at Harvard University.

Do you have a gene of interest but have run into a wall trying to study it? It happens. Is it an evolutionarily conserved gene? Can you find an ortholog in the Drosophila genome? Continue reading and I’ll show you how Drosophila can be used to push your research in new and exciting directions.

Drosophila are very easy to manipulate genetically and have limited genetic redundancy (meaning, there’s more of a chance of seeing a phenotype since additional genes that can do the same function are less likely to exist). If there’s an ortholog of your favorite gene (YFG) in Drosophila (and even if there’s not!) the wealth of Drosophila genetic tools available allow you to study many aspects of your gene’s functional biology in a living organism. This is the first post in a three-part series. We’ll first discuss how to get started on fly work in this post. The second post will detail a major tool used by Drosophila geneticists (the Gal4/UAS system), and the third post will describe how you can make your own mutant flies.

Find Drosophila Resources at Addgene

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Topics: Lab Tips, Drosophila, Quick Guide to Drosophila

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