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Plasmids 101: Inducible Promoters

Posted by Mary Gearing on Jan 18, 2018 9:34:59 AM

Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters. like CMV, EF1A, and SV40 promoters, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In this post, we’ll discuss inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. Keep a look out for a future post where we’ll discuss repressible promoters.

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Topics: Lab Tips, Plasmids 101

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

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

DIY DNA Ladders from Penn State University

Posted by Beth Kenkel on Jul 14, 2017 10:30:00 AM

Two plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently deposited with Addgene. A team of undergraduate students led by Dr. Song Tan at Penn State developed the plasmids, pPSU1 and pPSU2. When restriction digested with PstI or EcoRV, these plasmids generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially available ladders, the 100 bp ladder works well for both agarose and native polyacrylamide gels.

Get Tips on Verifying Your Plasmid

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Topics: Hot Plasmids, Lab Tips

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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