...Dolan, F100Research, 2016). These events have also started progress towards solutions. The 2015 Boston FoR...landscape facing all scientists, both young and established, as they embark on or continue their careers ... upcoming implementation of the Federal Labor Standards Act (FLSA) which mandates that all workers across...graduate students who work as teaching or research assistants to join unions. The topic of unionization continues...problems facing postdocs and graduate students, and started to define solutions but this year, together with...of Howell Communications Philip Brenner, PhD - Assistant Professor of Sociology. University of Massachusetts...uses synthetic biology to create tools to better understand human disease.  Outside the lab, he is interested...

... whether a certain methylation state causes a condition. To understand mechanisms and, eventually, develop...’s dCas9… with no modifications). Ready to get started?   Figure 1: Break free from the ball ..., transcriptionally active dioxygenase  When I started to work on building a CRISPR/dCas9-based tool for...expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can be achieved...achieved by fluorescence-activated cell sorting, and stable gRNA expression can be achieved using lentiviral...backbones expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy...

...and methylated states. Thus, this system is a reliable tool in reporting methylation status of these sites...the headlines, with sotires like the potential devastation of the palm oil industry through epigenetic effects...grandchildren; in this study,epigenetic changes were statistically significantly associated with reduced life-span...syndrome is characterized by low muscle tone, short stature, cognitive disabilities, problem behaviors, and...molecular biologic techniques to further the understanding of epigenetics; techniques include chromatin...Revolution: How Modern Biology is Rewriting Our Understanding of Genetics, Disease and Inheritance Amazon ...

... For anyone just starting to use flow cytometry, it’s generally recommended to start compensating with... depending on your flow cytometer’s setup. For instance, BV510 and AmCyan have almost identical emission...needing to use your single-color controls as a starting point. Applying compensation to your experimental...your main population and singlets. Then, you can start to compensate on your experimental samples. Let'... what two markers your fluorescent markers are staining for. If a cell is positive for both markers, it...challenging! But we hope this guide can help you get started. Remember, you can always change your compensation...

...use in the lab? Take for instance, the commonly used Myc-tag. Who first started using it in protein tagging...larger than the substance P peptide, but still quite small. However, unlike the substance P tag, they found...experiment involved a peptide from the neuropeptide substance P. This protein had a highly specific antibody...meaning the tag could be quite small. However, substance P has an amide group added to its C-terminus post-translationally...future. These newer findings simply add to our understanding of how this antibody works and allow users to...

...applied to PhD programs in Biology around the United States in 2021, I know that this process only became even... even more complicated with COVID-19 and standards for hybrid, virtual, and in-person events change seemingly... I reach out to potential mentors?” “Where do I start with picking universities?” and “How does COVID-...prospective student of such-and-such university State how you found their lab (usually through the university...other. Before COVID-19, financial and logistical obstacles could make touring potential programs and universities...online meetings with current students, faculty, and staff have become a new way to allow you to get important...options are very handy in moments like that! For instance, during my application cycle, two schools on opposite...

...cells don't stay out too long While you’re preparing things, make sure your cells don’t stay out too long... on the hood for at least 15 minutes before you start any tissue culture work and make sure that clean...as well as that watch you’re wearing before you start. Also, avoid unnecessary talking because talking... the hood and can wipe them all down before you start. This will also help you avoid moving things in ...already be at 37 °C. Most labs have a water bath constantly at 37°C for this purpose.Learn about the effects...split very soon. There is power in numbers! When starting off with a new cell line, grow the cells, aliquot...

...cancer-related complications each year. Despite these grim statistics, new strategies for attacking cancer are yielding...the circulatory or lymph systems to spread, or metastasize. Many cancerous cells will die trying, as the...technologies become available almost daily, yet obstacles remain. One of the biggest challenges facing researchers...project continues to evolve rapidly and, with assistance from the research community, will soon expand...pathways with potential to confer anticancer drug resistance." Science signaling 7.357 (2014): ra121. PubMed... Resources National Cancer Institute: Cancer Statistics Page National Cancer Institute: Ras Pathway v2.0...Resources on the Addgene Blog Tackling Cancers' Drug Resistance with a New Screening Kit Learn How the Clon Tracer...

...plasmids that need extra stability, such as some of our viral vectors, in NEB Stable cells. Resource information...in the example above that the bread crumbs trail starts in the browse page, continues through the depositing...Addgene Sequences’).  These sequences are provided in FASTA format (example) and can be analyzed using Addgene...Addgene’s single plasmids are sent as bacterial stabs at room temperature. Core of the plasmid page - ...the plasmid in your experiments: Backbone: The starting vector that the depositing lab modified to create...is shipped. Copy number (if known), antibiotic resistance, and optimal growth temperature are listed, as...

... a selection marker to generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA ...experiments, the following framework can help get you started: Determine what type of outcome you are trying...expression system and delivery method: Do you need stable integration or is transient expression sufficient... Likely contains a selection marker to generate stable cell lines. Packaging and Envelope plasmids provide...necessary components to make lentiviral particles. Stable, tunable expression of Cas9 and/or gRNA in a wide...and used to generate AAV particles. Transient or stable expression of SaCas9 and/or gRNA. Infects dividing...

... Ac5-STABLE2-neo T2A Insect   For the co-expression of multiple unique genes, you can start with a...they are useful, how they work, and how to get started with them. Why Use multicistronic vectors? Detecting... a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to easily screen...added to the end, while the downstream cistron will start with the Proline.  The table below lists the four...peptide to improve cleavage efficiency. How do I get started? If you are looking to co-express your gene of ... protein or selectable marker, it is easiest to start with a plasmid that already has the multicistronic...

...Drew Endy's lab at Stanford develops engineered DNA systems capable of storing data and computing inside...challenge. We have a strategy that organizes how we start the work, and it begins with the central dogma -...beginnings of an operating system for programming life, starting with the central dogma. Addgene: And the BIL gates...these parts be made freely available? Endy: When we start thinking about languages, humans have two types ...into practice? Endy: I think it's important to understand that these platforms, or pieces of a platform...incredible is the bioeconomy is already pretty substantial. It's estimated that genetic engineering as applied... al. "Precise and reliable gene expression via standard transcription and translation initiation." Nature...

...situation is already complex enough that a deep understanding of retroviral biology is necessary in order ...order to use the vectors correctly. A solid understanding of the structure of a wild-type viral particle —...normal, replicative life cycle — can help you understand these seemingly odd choices and lay the foundation...aspects of the viral “life cycle” in order to understand what these vector particles do once they come... thanks to Addgenie Rebecca Hutcheson for her assistance with lentiviral replication.  Resources and ...Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives. Viruses, 13(7), 1288. https://... the National Academy of Sciences of the United States of America, 54(1), 137–144. https://doi.org/10.1073...

...research. Understanding the depth and breadth of this bias, and then using that understanding to intentionally...Studying ancestral group dependencies  Take, for instance, a recent preprint released by the Boehm and Beroukhim... Aaron knew that their study had significant statistical limitations, and expected to find only a few,.... Like any diligent scientist, they sought to understand these strange results by digging deeply into ..., it primarily reflects European ancestry.  Understanding the impact Fig. 3: Histograms indicating...broader swath of human genetic diversity, to better understand how the use of a primarily European reference...

...kits. Kit-free alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid... DNA gel extraction with glass syringe filters Starting material: bacterial cultures or agarose gel slices...capture up to 150 ug of DNA, and filters can be stacked together in series to increase binding capacity...commercial kits.  DNA gel extraction with glass milk Starting material: agarose gel slices Product: DNA fragments... enzymes.  Cellulose nucleic acid purification Starting material: plant, microbe, blood, or mammalian ...Regeneration of silica columns for DNA purification Starting material: PCR product or agarose gel slices Product... the National Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073...

...libraries performance via the depletion of a gold standard set of genes (1,580 essential and 927 non-essential...effective in a narrow range around the transcription start site (TSS). The Root and Doench lab selected sgRNAs... selected and ranked based on the previously established sgRNA rules and  potential on- and off-target...al. demonstrated that the Calabrese library substantially identified more hits than the SAM library approach... a comparison study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic...reading frame (ORF) overexpression libraries for resistance to MEK inhibition. Although both Calabrese and...allow scientists to conduct genome-wide screens in standard cultured cells as well as more difficult conditions...

... retinal gene therapy. The year was 2001 and I started a job as a technician in a lab using adeno-associated...hemophilia, muscular dystrophy)? AAV gene therapy started in the mid-90's with a lot of work and a little... luck A pioneer in the field, Dr. Jean Bennett started working on ways to deliver genes to correct genetic... another 6 years before the 1st clinical trial started, and 10 more years to obtain FDA approval and make...an ever-growing number of clinical trials for devastating genetic diseases such as hemophilia, muscular... first clinical study combining these tools may start as early as next year. Twenty years after falling...Naldini,L., 2015. Gene Therapy returns to centre stage. Nature Vol 526, p.351. PubMed PMID: 26469046. 6...

...storage and distribution at Addgene are DH5α, NEB stable, ccdb survival, and Pir1. The majority of plasmids...plasmids contain either an ampicillin or kanamycin resistance gene. Every step along the way involves good ...and prepare fresh media with antibiotics at each stage. The barcodes and LIMS help us track each sample...detail-oriented lab team that works carefully to catch mistakes. Each sample that is transformed is plated onto... way that can be easily traced. If you’re just starting a lab, or are looking for a way to organize samples...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many forms...with a barcode reader and an Excel spreadsheet. Start by scanning the sample barcodes into the cells in...

...purification of recombinant proteins. Tags for stability and solubility What are some of the hurdles to...protect against proteolysis. That’s one form of instability. Prokaryotes can also have a hard time folding...s not folded correctly, there’s no point in crystallizing it or testing its function. Small ubiquitin-...ubiquitin-related modifier (SUMO) can help with folding and stabilization, as can maltose-binding protein (MBP). Overexpression... site GST Large Protein 26 Purification and Stability Good for purification with glutathione; protects... detection SUMO ~100 amino acid protein  12 Stability At N-terminus, promotes folding and structural..., with various mutations used to increase its stability activity, can be readily purified using plasmids...

...the signal intensity of the sample to that of a standard curve to extrapolate the concentration of antigen...chromogenic substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and...sample and is calculated by extrapolating from the standard curve. Because the primary antibody is directly...ELISAs generally have higher levels of background staining. Antigen-containing samples often include undesirable...non-specifically to assay components and increase background staining. Direct ELISAs also lack the signal amplification...sample and is calculated by extrapolating from the standard curve. Scientists often opt for sandwich ELISAs...cross-reactivity Higher level of background stainingLess sensitivityLess specificityLimited availability...