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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: Lab Tips, Techniques

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

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