...light and chemicals respectively. Didem Goz Aytruk from Connie Cepko’s Lab at Harvard Medical School describes...although fluorescent proteins are incredibly useful, chemical fluorophores can still be used in a wide variety...variety of experiments. Read more to find chemical fluorophores for your next experiment. Nucleic acid based...systems that report the order in which specific chemical inputs are received by a cell. These so-called...report upon and/or engineer “temporally ordered biochemical events” such as those that occur during development...containing only the sequences required for viral replication and transcription as well as reporter genes. ...Membrane-Rupturing Microscopic Tools In this post, Jessica Polka, formerly a postdoctoral researcher in the...

...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. ...absolute quantification of viable lactic acid bacteria in faecal samples." Journal of microbiological methods... we have used quantitative PCR to evaluate the physical titer of our preps. We often repeat this assay...factor of 2. This means that if you set up two identical assays with the same sample, you could end up ...would be valid. This is why the AAV reference is critical. It allows you to determine if your titers are.... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...detection of the product. As an added benefit, replicates are built into the technology. One well (containing...

...many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to... but this research is still in the pre-clinical/early clinical stages. Researchers are also working to...to learn more about the current (and future) applications of lentiviral vectors! Lentiviral vector history...original system, the possibility of creating a replication-competent virus via recombination between the...of recombination events necessary to create a replication-competent virus. Second, Tat is eliminated through...barcoding libraries have been used for complex applications like lineage tracing in heterogeneous tumors...2015: lentiCRISPR v2, used both for targeted modification and genome-wide screening.   Lentiviral vectors...

...terms of biosafety? A8: According to the American Biological Safety Association, "the two major risks ...lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown... looking for some additional information or clarification on these vectors. Read on to find the answers...scientists often refer to as “retroviruses” are technically gamma-retroviruses - another, separate member...differences may seem subtle they result in a key physiological difference between lenti- and retroviruses. ...transfer vectors for transient transfections? A2: Technically, yes. Will it actually work? That depends. The...when using a plasmid backbone that was designed specifically for transient expression (find some useful backbones...

...this approach has been adapted for the identification of physical materials that we use in our labs, including...RRIDs and the Resource Identification Initiative Compact identifiers for biomedical data How Addgene works...about which reagents were used? Unambiguous identification of a reagent is crucial for reproducibility...to identify and reference documents, files, or physical reagents. What are persistent identifiers? Perhaps...persistent and exist online indefinitely. PIDs historically referred to objects that exist online, but this...item. Given a specific Addgene ID # listed in a publication, you can quickly pull up the associated plasmid...plasmid page also helps researchers find other publications that have used the same plasmid through the ...

... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for...Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these... their bacterial counterparts, including how replication occurs and whether selection is necessary for... way. Selection methods, described below, are typically employed when creating stable cell lines and are...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus...antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40...

...approach that has been the historical standard. Specifically, in our recent publications, we used the cancer ...result, how most of these “variants of unknown significance” (sometimes called “VUS”) function is unknown... if the majority of information on each cancer clinical sequencing report includes rare variants that ... variants across a large number of genes as systematically as possible. In developing these assays, we...tumorigenesis assays, drug epistasis assays, and morphological profiling can be used as generalizable strategies... are described in this paper Systematic morphological profiling of human gene and allele function ...pathway reference collection is described in this publication We also wrote an extensive review on the exciting...

...the amount of a given factor within a biological system. Basically, biosensors provide quantifiable readouts...biosensor?! You can do more than just ‘sense’ biological compounds and reactions; you can quantify them...of the biosensors used in research are very sophisticated, but at their core they can all be boiled down...pretty simply – they are systems that measure biological phenomena by generating an output which is directly...readouts for levels of different chemicals, hormones, proteins, etc., that you are interested in. The most...we will refer to as the analyte, needs to be specifically recognized by another factor, the receptor. ...fluorescence, pH, etc.). The transducer will generate a numerical value which represents the amount of analyte present...

...intended application - Remember, just because your antibody performs as expected in one application does ...why validation is so important. Antibodies are a critical component of the researcher toolkit due to their...major culprits in the reproducibility crisis of biomedical research (Baker, 2015; Bradbury & Plükthorn, ...strategies for antibody validation in different applications. Here, I want to highlight the general suggestions...intentionally generalizable and not specific to certain applications or antibody targets. That said, not every strategy...will be suitable for every antibody or every application. The goal is to show that your antibody has been...strategy to use will depend on your intended application and available resources.     Figure 1...

...their visions for a successful and sustainable biomedical research sector at our first Future of Research...discussed, many related to the structure of the biomedical workforce. Quotes taken directly from participants...of Medicine and the University of Massachusetts Medical School. Graduate education programs are also beginning...internship to be taken in an industry, regulatory, or clinical environment. It remains contested, however, whether...shape and inform the overall structure of the biomedical research sector, and what a tenable workforce...The panel discussion on Friday morning will be dedicated to Academic data and the Labor Market. On this...Shapes Science’ and contributing to the NIH’s Biomedical Research Workforce Working Group. Along with ...

...Democrats more often than Republicans, the March is not about Democrats or Republicans (16). This is a Pro-...general (1). Science and politics? Science is an apolitical process for seeking knowledge. The process begins... again. No part of this process is inherently political so why is there a march in Washington, D.C., the...States government? Politics and science are intrinsically linked: governments exist in reality and science...as evidence of climate change, the safety of genetically modified organisms (GMOs), the absence of a link... grant in the last five years as polled in the American Association for the Advancement of Science) believe... it.  3) Linking scientists to one particular political party while alienating the other While scientists...

...conflict is not always what was communicated but how it was communicated. The next step is to come up with...of lapsed communication. Making some assumptions about how something should be communicated is necessary...set of transferable skills, coupled with your technical skills, can open new career opportunities for ...conflict can also be good. In science, a reviewer’s critical comments on your manuscript is a form of (hopefully...scientist. And some amount of conflict is actually critical in the business world. Companies and organizations...traced back to one of a few trouble spots:  Communication (or lack thereof): Information sharing gets ...organization gets, and creating new avenues of communication can create as many problems as it can solve....

...well as protease sites to facilitate protein purification. We hope this kit will allow you to quickly ...for a variety of synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you...polycistronic E. coli expression constructs. Some applications of plasmids from this kit include the production...: Čelešnik, Helena, et al. "Biosafety of biotechnologically important microalgae: intrinsic suicide switch... Rogers, Jameson K., and George M. Church. "Genetically encoded sensors enable real-time observation ...and Charles E. Melançon III. "Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics... Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri system The Gomis-Ruth...

... its nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being...characterizing the 4D nucleome. Future modifications and applications Ma et al. developed this technique ...plasmids from this publication at Addgene. A protein-tagging system for signal amplification in gene expression...technique published every week! One of the newest applications is a colorful system that uses fluorescently... The technique successfully resolved loci with physical map distances of 75 and 2 Mbp, with the calculated...distances correlating with the previously established physical map. In comparing pairs of targets ~2 Mbp apart... et al. anticipate that the technique will be applicable to studies of cell cycle progression, epigenetics...

...show all publications by an author, total number of citations, and the number of publications that heavily... staying on top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search...read the abstract or full text of the paper, publication date, a relevance score, and type of article....that term. You can also set up alerts for new publications from a particular author. Saving papers Papers...interest.  Best uses Accessing the vast body of biomedical research papers. Twitter Scientists often use... work, so it’s also a good place to look for publications. Bonus: papers have to be summarized in 280 ...organizations, and universities often tweet about publications in the fields of research they support. Monitoring...

...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may... a strong signal and to work in a variety of applications. The desired specificity of your secondary antibody...affect my experiment?” Well, it depends on the application and target. If you are running a basic western...target is expressed at very low levels or your application requires a high degree of affinity, then species...considered when choosing an antibody. In some applications probing species-on-species, i.e. using a mouse...choosing antibodies   Complexity is a key factor Biological systems tend to be complex with a variety of ...

...et. al.). Physical versus functional titer Viral titers usually come in two flavors, physical or functional...the target cell’s genome. Physical titer The two most common assays for physical titer are direct p24 measurements...functional. Physical titers measure the amount of viral particles in a sample and are usually based on the...produced can actually infect cells. These assays typically involve infecting a target cell line with your...particle. Consequently, titers based off of p24 quantification tend to be overestimates since they can include...quantify the amount of PCR product that comes from amplification of the original plasmid which can over-inflate...methods tend to be more accurate than measuring physical titer, there is a risk that they may underestimate...

... offers a significant improvement and simplification of the original technique, specifically in terms ...interaction with limited biological materials. Problems with Chip-seq ChIP-seq typically requires a large amount...material for a ChIP-seq experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking during...interactions for subsequent sheering of DNA by sonification. Though it helps capture weaker or more transient...Washington. CUT&RUN doesn’t require fixation and significantly decreases background noise, reducing the read...development using the Drosophila wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings...dissections. With CUT&RUN, we have had success with significantly fewer wings, such that enough tissue for an ...

...improving expression of nutritionally and therapeutically significant proteins. In this project she aims to...pests. By developing biological agents such as insecticidal proteins, the use of chemical pesticides may be...scientists work on projects spanning from the identification of plant compounds and insecticidal proteins...get tested for their potential agricultural and medical use. Find More Optogenetic Tools Like pDawn  Making...bacteria. Learn more about the different CRISPR applications in plants.     Transgenic yeast as source of... yeast capable of desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc...one of the reaction’s metabolites. One useful genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which...

...techniques in the biological sciences and constitute foundational elements of biomedical research. Traditional...Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation...Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration... multi-step process consisting of digestion, purification, ligation, and transformation. While restriction...sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive time requirements...is ligation independent, it does not require purification of insert or vector products, nor does it require...Digestion products are then transformed directly into chemically competent E. coli and transformants selected ...

...Attempts to remove excess bone surgically cause the body to “heal” the surgical site with even more bone that...therapeutic biologic design (ADCs) along with pre-clinical analytical development. His areas of expertise include...protein engineer currently working at Sanofi Pharmaceuticals in Cambridge, MA. When I joined Sanofi/Genzyme...to get in shape. After three kids, I had put on typical middle age weight and “exercise” consisted of taking...named AJ Gonzales who suffers from Fibrodysplasia ossificans progressiva (AKA FOP). This terrible disease ... But, there is HOPE. FOP is a genetic disease typically caused by a single R206H point mutation on the...growth in mouse models. As this biologic moves into clinical trials, there is new HOPE for AJ. But, as we in...

...the gut side of things, the authors optigenetically and chemically excited a class of enteroendocrine ...behavior, or the animals being monitored were physically hooked up to invasive, cumbersome systems during...and stimulate or manipulate responses through neurological means like electric stimulation, drug administration...programmable microcontroller, NeuroStack coordinates optical stimulation of its associated fibers across multiple...fibers. As important as its capabilities are the practical considerations of actually using it - the device...to mCherry to the brain tissue. They performed optical stimulation using NeuroStack and the fibers as ...as previously described, then measured electrophysiological potential afterwards. This measurement trended...

... This allows them to replicate viral plasmids with the SV40 origin of replication. To generate a virus...proteins at high levels. In a case where clinical applications of recombinant proteins are necessary, HEKs...serum-free growth, which significantly reduces the cost of production. Research applications There are two areas...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent...cell biology, there are thankfully many other applications that they are useful for! What the HEK is it... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...

...Scientific Publication, where diverse approaches to peer review were discussed. Science communication opportunities...developmentally programmed regulation of DNA replication and gene transcription during vertebrate embryogenesis...with people, nurturing networks, as much as with technical knowledge. I leverage a breadth of knowledge to...I am constantly teaching – often explaining a technical piece of research to others outside that specialty...and jobs. At Science, which is published by the American Association for the Advancement of Science (AAAS...punctuation can be wildly funny as well as financially critical. Those who work on the publishing side of science...There is a rich and active community of science communication professionals who are thinking about, for example...

...BglBricks: A flexible standard for biological part assembly. Journal of biological engineering. 2010;4(1):1. Pubmed... diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput...elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...transgene expression, or a variety of other clever applications. In one example, modular constructs generated...methods are suited for particular downstream applications. The tradeoffs between various cloning methods...

... also be perturbed by supraphysiological expression levels. BiFC quantification This is where BiFC outshines...Miller-Rhodes from the University of Rochester Medical Center.  You’ve probably heard of Forster Resonance...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...2018), the generation of such resources in less genetically tractable organisms lags behind. BiFC can also...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described...to the N- and C-terminal end of each PIP and empirically test which combination provides the best fluorescent...

...biology. Therefore, my lab is dedicated to development of new biological technologies. The locus-specific...been working on development of technologies for biochemical analysis of genome functions such as transcription...technology, which uses locus-tagging and affinity purification, in 2009. However, iChIP requires insertion ... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic...describe this technology and some of its potential applications? Fujii: We generated a series of retroviral ...researchers to use enChIP in a wide variety of biological systems. We are currently developing several ...interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences. Thus, we've made ...

...to directly activate and inactivate biological processes. Chemical effectors are suboptimal for a number...neuroscience. In fact, precise light-based control of biological processes is highly useful in other fields, including...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can... also be used to engineer new pathways with sophisticated gene expression controls. Most cell types do...system to develop a bacterial camera that prints a chemical image, as well as a genetic method for the computational...light; these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...; only two plasmids are required per system. Applications of these systems The large dynamic range of ...

...Luciola italica)Southern Russian firefly(Luciola mingrelica) 562 64 Luciferin ATP No Jamaican click beetle...Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting in the...gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection... is measured resulting from the catalysis of a chemical reaction requiring luciferin, ATP and oxygen as...Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity laser...Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon...Background Lower Higher Post-translational Modification Not Required Required Photobleaching/Phototoxicity...

...antibody interaction and/or the downstream application. Typically, if the target protein will be detected...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Immunoprecipitation Overview An immunoprecipitation reaction is typically carried out in one of two ways. Both methods utilize.... 2) An antibody immobilized on a bead binds specifically to the blue triangle protein but not the other...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this... or magnetic beads with commercially available chemical agents. Directly conjugating the primary antibody...interactions To prepare for an IP, samples are typically lysed in a non-denaturing buffer containing non-ionic...

...fields where communication and writing are key skills. Other fields, often highly technical or skills-based...proactively address questions about your application. These could be logistical, like briefly explaining a gap ...excellent communication skills” try giving an example, like “I have extensive communication experience...usually yes! A good cover letter won’t hurt your application, but it can certainly help you stand out.  In...consider cover letters an important part of the application. Even within an organization, there can be variance...good cover letter!) As with any part of the job application process, it's a good idea to talk to people in...work on [phenomenon X], I read several of your publications on [specific topic]. I am extremely interested...

...and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then...Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting was performed...transduced with Cas9 expressing lentivirus at the indicated multiplicity of infection (MOI). Anti-vinculin...lines generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and...need to use FACS (like they did in the original publication). I thought I could see GFP expression with a...

...revolves around synthetic humans called replicants which are genetically engineered by a powerful corporation...fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the prototypical base editor...genetic code of living organisms and its societal implications even before the discovery of genes themselves...explores the concepts of animal testing and modifications. The premise of the novel involves Dr. Moreau...technology led to the development of many important medical and agricultural products. But it also made SciFi...2018) were highly successful in the box office, indicating the insatiable appetite of the moviegoers for...a theme park.  Released in 1997, GATTACA is an American SciFi film starring Ethan Hawke, Uma Thurman and...

...prior publications to go on, a submitted manuscript could only have been viewed as a significant improvement...causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA)...the piñata builders - the reviewers. After some physical exertion on the part of the party goers, the piñata... solutions that facilitate most experimental applications in CRISPR engineering. When compared to that...easy to fault the lack of features in a single application. The question is then, should we use all tools...those engaging in CRISPR design, but it is a significant hurdle made worse by an ever increasing marketplace...the other hand some software tools make use of empirically derived and/or prior knowledge to inform on the...

...natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is...recognition and cleavage. This sequence flexibility indicates that gRNAs could be incorporated into these constructs...the 5’ end. PTG construct schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage...) How many loci can Cas9 modify? To test the application of this system to gRNA multiplexing, Xie et al...deletion with 4-20% frequency at the four loci, indicating that Cas9 can be guided to at least 8 distinct...multiplexing further illustrates the potential of PTGs. Applications of PTGs The major advantage of the PTG system.... Though the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely...

...Stanford University. Sex and gender are critical components of biological research that are often forgotten ...provide a table indicating the sex of the most commonly used cell lines appearing in American Journal of Physiology-Cell...the past decade. For more practical help, there are numerous methodological articles to consider—we have... that of human clinical trials. And, as Janine Clayton points out, late-stage clinical studies that draw...effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to why... disease being treated, and other hormonal, immunological, and environmental factors. How to include sex... (NIH) now requires that sex be included as a biological variable in all NIH-funded research. Janine Clayton...

...current CRISPR research focuses on its potential applications for human medicine (Waltz, 2016), the potential...engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a ...commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species including ...engineering using CRISPR, highlight the specific modifications to CRISPR machinery that allow for the use of...CRISPR principles). However, plant-specific modifications to commonly used CRISPR plasmids are necessary...selected the correct CRISPR components for your application, it is time to deliver these components to your...detergent, Polyethylene Glycol (PEG), although the application of this approach is limited to protoplast cells...

...proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation patterns...Depending on the research question and downstream application, one or several of those techniques can be used...populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...cell membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane...intracellular labeling with antibodies requires chemically pre-treating the cells to allow for entry of ...but preserving the cellular state (think of mummification). Permeabilization, as the name suggests, perforates...

...exclude duplicate events, and the Live/Dead gate to focus your analysis on what’s biologically active....exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably..., you’ll first need to learn a little bit of theoretical background on the different parameters that your...and W describe the shape of a histogram, which graphically represents the time and intensity of the cell... fully depend on your individual experiment. Practical example: lymphocytes in a mouse tumor sample You... You made it! So much about the theoretical background. Let’s dive into the praxis of gating strategies...show up in a straight line one the plot, while duplicates deviate from that pattern. A.                                                                                                         ...

...normalization and loading controls needed for relative quantification of a western blot — and why you might want ...accurately compare different lanes? In relative quantification, the POI bands are normalized to the loading...other errors in experimental design and gel quantification. Figure 3: In a perfect world, protein...On this gel, the protein of interest band is significantly lighter in “condition 1” than “condition 2.”...and it’s also problematic for computational quantification. Outside the linear detection range, band intensity...common error associated with Western blotting quantification” (Gilda & Gomes, 2013). To address this source... Gilda & Gomes, 2013. Figure 7: Semi-quantification of liver lysate using different loading controls...