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CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

Posted by Guest Blogger on Feb 13, 2018 9:51:55 AM

This post was contributed by guest blogger Matthew J. Niederhuber, a graduate student at UNC Chapel Hill.

Chromatin immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where a protein binds genome-wide, and has been widely applied in many model organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations. The enzyme-based CUT&RUN method overcomes many of these limitations and makes it easier for you to map protein-DNA interaction with limited biological materials.

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Topics: Plasmid Technology, Techniques

In Vivo Biotinylation of Bacterial Fusion Proteins

Posted by Guest Blogger on Jan 25, 2018 9:09:35 AM

This post was contributed by guest blogger Jon Backstrom, a biochemist in the Vanderbilt Eye Institute and Tonia Rex's lab.

A common strategy to determine the binding kinetics of a purified protein involves immobilization on a solid support. This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on a reduced glutathione matrix. The advantage of a fusion protein is the efficient purification of an already immobilized target protein. The disadvantage is that the GST moiety, which forms dimers, may influence binding kinetics of the target ligand. Another important consideration is whether the affinity of an experimental protein-ligand interaction approaches that of GST-glutathione.

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Topics: Plasmid Technology, Hot Plasmids, Techniques

CAPTURE-ing Chromatin Interactions: Using CRISPR-dCas9 to Study Gene Regulation

Posted by Beth Kenkel on Dec 7, 2017 9:16:43 AM

Plasmids can be amazing and simple tools for studying gene regulation. They are used to study how transcription factors and other trans-regulatory elements (TREs) and some cis-regulatory elements (CREs), like promoters, influence gene expression. However, scientists frequently return to native chromosomes because chromatin context matters. The impact of TREs and CREs on gene expression is commonly investigated via Chromatin Immunoprecipitation (ChIP) and chromatin capture techniques, respectively, but these two separate methods are not without their own technical challenges. Enter the Xu Lab's CAPTURE, a method for identifying TREs and CREs that partners CRISPR’s targeting abilities with the strength of the biotin-streptavidin interaction. CAPTURE is capable of identifying old and new TREs and CREs, CRE-CRE interactions, and has even provided enough data for Liu et al to re-draw the beta-globin locus regulation model. Read on to learn more about this captivating tool!

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Topics: CRISPR, Techniques

Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

Posted by Guest Blogger on Oct 19, 2017 9:54:49 AM

This post was contributed by guest blogger Katrin Michel.

Cre-lox is an incredibly popular and powerful site specific recombinase (SSR) system, but it only gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated possibilities for site specific (and often cell type specific) control of DNA recombination and gene expression can be advanced by the coordinated use of fellow SSR system FLP-FRT. In addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations of FLP and Cre enable additional levels of genetic control.

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Topics: Plasmid Technology, Cre-lox, Techniques

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

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