We narrowed to 858 results for: CAL;

...are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV genomes...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...genomes/mL) can be measured by: Optical density: particle concentration is measured by looking at absorbance...260nm corresponds to 1.1 x 1012 particles/mL. To calculate the particle number: OD260 reading x dilution ... days later. Plaques are counted and titer is calculated in terms of plaque forming units (pfu), taking...1236. PubMed PMID: 17546019. 2. He TC. et al. A practical guide for using the AdEasy System. 3. Jager, Lorenz...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...modifications at the DNA level in a temporally and locally defined manner. This allows scientists to draw ...and not targeted to a specific area. Some other technical considerations for stereotaxic injections are:...be high enough to maximize possible outcomes. Typically, AAVs have to be minimum around 1012-1013 vg/ml...has to be large enough to target larger areas, typically a few hundred nl. Alternatively, for larger brain...expression levels of the viral vector trangenes typically occur two weeks after injection. Therefore, it...

...embryos. But once you generate a growing colony of genetically engineered mice, what can you do? In this post...Cre-lox lines, and how to properly control for genetically engineered mice in your experiment. As you’ve...ve learned in Part 1, there are many types of genetically engineered mice: transgenic mice, knockin and...getting a small piece of tissue from each mouse (typically via either a blood draw, tail clip, or toe clip...Hemizygous mice can be included in breeding strategies identical to those used for breeding heterozygous mice. ... cases Whether your mouse colony consists of genetically engineered mice with a single mutation or strains...prepared to plan an experimental strategy that uses genetically engineered mice. Best wishes on your experiment...

...generated in vitro using synthetic genes that are typically expressed from a plasmid or from an integrated...share, making recombinant antibodies the most practical choice. Hybridomas are much tougher to ship, often... often lack sequence data, and may genetically drift over time. Antibodies made from animals are limited...the ease with which scientists can now improve genetically-encoded research tools, it is shocking that the...Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531–533 . https:/...Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting recombinant mouse monoclonal...for the Selection of Monoclonal Antibodies for Clinical Use. In: Antibody Engineering. InTech Additional...

...mass spectrometry (AP-MS), lack the ability to specifically identify proteins that interact with a particular...lysed before the bait protein is pulled down, typically with an antibody. This can artificially select...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA...functional BirA* enzyme. This type of experiment is called a protein fragmentation complementation assay (... via Western blot, and that result in proper localization of the fusion protein. Advantages of Split-BioID...Further characterization of these candidates is critical to understanding how they interact with the bait...Varnaitė, R. (2016). Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling...

...likely that the shifts caused to the practical and philosophical landscape of the scientific enterprise...precipitated increases to the NIH recommended pay scale – used by many institutions to set postdoc wages...focus toward advocacy and action with sessions specifically chosen to better define productive routes for...Riglar. David T Riglar is a postdoc at Harvard Medical School. In the lab, he uses synthetic biology to...References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research from its systemic flaws." Proceedings...Pubmed Central PMCID: PMC4304227. 4. Revitalizing biomedical research: recommendations from the Future of ...

...What’s behind these sights are an abundance of biological molecules like chromoproteins, fluorescent proteins...proteins and luminescence have been widely used in biological research for a long time, chromoproteins are ...just now being developed for a broad range of biological inquiries.  What is a chromoprotein and how ...photosynthetic activity. This still allows photosynthetically active wavelengths to reach zooxanthellae...the Sea AnemoneCnidopus Japonicus. Journal of Biological Chemistry 281:37813–37819 . https://doi.org/10.1074...Pigments of Pocilloporid and Acroporid Corals. The Biological Bulletin 189:288–297 . https://doi.org/10.2307...chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036...

...such small amounts of DNA (often on the picogram scale) that most quality issues are negligible. That said...small amount of DNA (in the nanograms to micrograms scale depending on the experiment), but issues with DNA... large amounts of DNA (easily in the micrograms scale and up). But perhaps more importantly, the DNA needs...the clean-up stage, plasmid purification kits typically use one of two common approaches — columns or ... result in incomplete lysis. You may be able to scale the amount of buffers used to ensure proper lysis...appropriate amount of beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep...and Milllipore Sigma’s GenElute™ kits were all specifically designed with environmental sustainability in...

...reverse-transcribed piece of DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (like HIV-1) — that have been ...(Prussin et al., 2015), only a small fraction typically cause disease. The gamma-retroviruses have their...transduction by vectors (which we sometimes still call “infection” as a shorthand, although it is not quite... proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128...

...candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic ...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons...few important details. For transposons to be specifically recognized during transposition, genetic cargos...Additionally, these transposon also exhibit something called target immunity, whereby multiple proximal insertions...Leveraging a unique marriage between distinct biological elements, CRISPR-transposons excel at programmable...candidate in the Sternberg Lab at Columbia University Medical Center. He currently studies CRISPR biology and...

...kind of biological framework, suggested to them that their findings may be the result of a technical artifact...Biomedical research has a long and well-documented history of racial bias, which still impacts and influences..., by extension, race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to ... Aaron knew that their study had significant statistical limitations, and expected to find only a few,...Seeking an explanation  First, they looked for a biological explanation, a relationship between the genetic...far larger and more complex problem endemic to biomedical research: pervasive bias that is easy for researchers...

...purification (TAP) once referred specifically to a combo tag comprised of a calmodulin binding peptide (CBP), a...to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids 101...overexpress and purify proteins can face any number of technical challenges depending on their protein of interest...consider junk protein. Though there are a number of chemical and peptide-based proteosome inhibitors, glutathione... relatively small sequence of amino acids, is basically a molecular leash for your protein. If you’re ...want to combine a fluorophore with a tag that localizes your protein to the nucleus? Or you want multiple...

...Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul Blainey’s lab has...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This platform...compatible with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...one of the virus’s most abundant proteins and a critical component for viral genome packaging. Addgene ...Genetic modification of microglial immune cells is critical for central nervous system research; however, ...cells.      Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with...gene of interest, reducing the immunogenicity typically associated with viral vector-based therapies. ...

... prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our...larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell population...The size and composition of your library can drastically impact what conditions it needs to properly amplify...can transduce your cells.  While Addgene uses a physical titer (via ddPCR) to quality control our viral...include a 1.3 mL aliquot with mostlibrary requests specifically to allow scientists to perform functional titering... D., Sanjana, N. E., & Zhang, F. (2017). Genome-scale CRISPR-Cas9 knockout and transcriptional activation...Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR...

...results (i.e. mistakenly calling a gene a hit when really it’s not). In practical terms, this means that... approach for identifying genes involved in a biological process. These screens provide an alternative...cutting than full-length guides (19-20bp)?  Small-scale studies (Tsai et al., Fu et al.) suggest 17-19 bp...non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and 6,750 safe-targeting...ricin screen. When safe-targeting guides are used to call hits from the growth screen, p-values were less ...References 1. Morgens, David W., et al. "Genome-scale measurement of off-target activity using Cas9 toxicity...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Vincent and Niroshi Senaratne, from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Niroshi Senaratne and Ben Vincent from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...

...structures that are processed by a ribonuclease called Dicer. In a great simplification of the process...pri-miRNA, which is then processed by a complex called Drosha to pre-miRNA. miRNA plasmids are engineered...backbone Choosing the promoter Mature siRNAs typically range from 19–22 nucleotides and are 100% complementary...’t just use the mature sequence in a plasmid and call it a day. You need to include both sides of the ...for quick in vitro experiments. shRNA plasmids typically use RNA polymerase III promoters, which transcribe... versatile gene-silencing machine. Journal of Biological Chemistry, 284(27), 17897–17901. https://doi.... DICER: structure, function, and regulation. Biophysical Reviews, 13(6), 1081–1090. https://doi.org/10.1007...

... why you might want to be careful relying on so called “housekeeping proteins.” Fully quantifying a western... later steps in this chain (blue) to infer or calculate things about earlier ones. In practice, multiple...popular choices for loading controls. These are often called “housekeeping proteins” and are detected with an... will almost certainly be overloaded. One paper called this problem “the most common error associated ...Data is presented as mean ± SEM. Adapted from Analytical Biochemistry, vol 440, Gilda, J. E., & Gomes,...loading control to β-actin for Western blots. Analytical Biochemistry, 440(2), 186–188. https://doi.org...Antibodies 101: The Basics of Western Blotting Technical Design of a Western Blot Troubleshooting and Optimizing...

...populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on...of interest via FACS using negative selection is called dump gating. Let’s say we want to sort out all ...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 ...esterified dyes that can enter cells freely are typically used. Inside the cell, the ester group is cleaved...intracellular labeling protocol is necessary, requiring chemical treatment of the cells after surface labeling....Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...especially helpful. I set out to make something practical and accessible. If there's something you come ...the Janelia Research Campus of the Howard Hughes Medical Institute. His interests include the quality control...

...able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...intracellular locations using Cas9 and track the localization of the complex. Broccoli, the other green fluorescent...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...high-affinity complex for RNA labeling and tracking." ACS chemical biology 9.10 (2014): 2412-2420. PubMed PMID: 25101481...and directed evolution." Journal of the American Chemical Society 136.46 (2014): 16299-16308. PubMed PMID...di-GMP and cyclic AMP-GMP." Journal of the American Chemical Society 135.13 (2013): 4906-4909. PubMed PMID:...a photostable RNA–fluorophore complex." Nature chemical biology 13.11 (2017): 1187. PubMed PMID: 28945233...

...where you can start to identify which items aren’t critical to your research success. If you can get away ...resources to keep, and which ones to abandon. It’s critical to make these decisions in the following order... budget can help you compare the monetary and empirical value that helps you determine whether or not ...know how to decide which components are the most critical to your research, negotiations can be much easier...negotiating finances, instead of solely discussing numerical values, try to keep conversations focused on research...saving the institute time and energy that would typically be wasted going through some of the questions ...is your goal, then understanding your needs is critical to a successful negotiation and collaboration....

...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins....this approach. This study generated the first optically active saCas9 protein. Stop light! Photoactive...different approach. These researchers used a genetically encoded photocaging technique to insert a light-removable...light-removable protecting group, specifically a nitrobenzyl photocaged lysine (PCK), on the Cas9 protein...Hemphill et al. presented data showing that this genetically encoded, photocaged Cas9 system could silence...editing and transcription. Label genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged...

...it's usually more efficient than plasmid donor. Typical design is to have 50–80 bp of homology arms on ...changes (>100 bp insertions or deletions), we typically use plasmid donor, with two homology arms on each...transfection in cells like HEK, we use around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector...plasmids. For ssDNA oligo design, the Zhang lab typically uses around 100–150 bp total homology. The mutation... giving 50–75 bp homology arms. The Zhang lab typically use PAGE purified long oligos. For large changes...100 bp insertions or deletions), the Zhang lab typically uses a plasmid donor, with two homology arms on...like HEK, the Zhang lab uses around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector...

...deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-...-to-inosine (A-to-I) RNA editor called REPAIR by fusing catalytically inactivated RNA-targeting Cas13 ...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ...editor still retains A-to-I editing. This system is called RNA Editing for Specific C-to-U Exchange, or RESCUE...

...you’ve saved. Repurposing regulated medical waste Regulated medical waste (RMV), which we collect in those...scores products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...companies to obtain these labels! Reusing plastic Historically, scientists washed and reused materials such...flow cytometers. These fluidic machines handle biological samples and are cleaned between each use. You...those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental developed...

...Regulatory DNA function is typically inferred based on correlative biochemical characteristics and ectopic...Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint...for deletion screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even...investigator at Boston Children’s Hospital and Harvard Medical School. His lab studies the molecular genetics ...Matthew Canver is an MD/PhD student at Harvard Medical School in the laboratories of Stuart Orkin and ...

...Kristin Krukenberg and Jessica Polka from Harvard Medical School and a long list of supporters from all of...peer review and grant awards systems need to be radically overhauled.  Finally, the science publishing system...excellence to begin effecting change.  Panelists call for action Each of the speakers in the panel had...Director of the Office of Postdoctoral Affairs at UNC, called her presentation “Disequilibrium, Disillusion and...Associate Professor of Systems Biology at Harvard Medical School, titled her presentation “How to Train Your...and take action. More Reading: “Rescuing US biomedical research from its systemic flaws” by Bruce Alberts...

...target specific tissues, even when administered systemically, without causing disease? Recombinant adeno-...receptor (AAVR)2 Carboxypeptidase D (CPD), also called AAVR25   AAV9 Terminal N-linked galactose3...integrin3 AAV11 Carboxypeptidase D (CPD), also called AAVR25   1Wu, Miller, et al., 20062Pillay...genomic structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often ...receptors and tissue tropism. AAV5 AAV5 is the most genetically distinct AAV. Though AAV5 is one of many serotypes...Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle. Human...is required for gene transfer. The Journal of Biological Chemistry, 276(23), 20610–20616. https://doi....

...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal...promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity to... the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...deadCas activator/repressor experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets...effectiveness as a control. Since off-targets typically confound an experiment due to their location (...

...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature...Ganguli, S., & Deisseroth, K. (2019). Cortical layer-specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...

...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...varying ratios of recipient plasmid to insert. It is critical to set up a negative control ligation reaction...electro-competent cells instead of the more common chemically-competent cells. You should perform, at minimum...purifications, you generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic...

... then study where the protein is localized, and how its localization and expression may change across ...transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since... also affect protein activity, half-life, or localization, these concerns are greater with SunTag. v1 ...

...poster tubes (or as our baffled flight attendant called them, "long, skinny things") and generally exuding... tales of back country skiing, fly fishing, and local grizzly bear attacks. He described one such recent...All Addgene depositors! Addgene was mentioned specifically in Dr. Zhang's introduction. His willingness...Cas9 that is improving the nuclease specificity dramatically and reducing off-target effects. Other young...discussed some of the many challenges of creating biological circuits, and I can only say this science is ... deposited and distributed – have changed so dramatically in the past 4 years. I kept busy for the whole...

...an antibody that works for your application, specifically detects your protein, is species compatible,...along with some pros and cons. FLAG FLAG is a synthetically derived 8 amino acid tag (DYKDDDDK) that can...possibly interfere with protein function and localization. If you need monoclonal GFP antibodies or guidance... N or C terminus of a protein may disrupt the biological function of some proteins. Protein folding issues... In rare cases, tags can also affect protein localization and solubility, so be on the lookout for aggregation...Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol...

...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...science research all the way up to the production of clinical viral therapies. Protein production  HEKs are...recombinant proteins at high levels. In a case where clinical applications of recombinant proteins are necessary...also grow in suspension, which allows for large scalability, and can also be adapted for serum-free growth..., which can make for a seamless transition of biological tools to other researchers using the cell line...

...Extremophiles - phylogenetically diverse microbes living in extreme physical and chemical conditions like...Stanford University became interested in exploring biological dark matter – large tracts of the microbial tree... to be diversity hotspots containing abundant biological dark matter and so the lab organized a sampling...like YNP thermal sites - are also of practical interest since they’ve previously yielded valuable enzymes...

...arguing with me. First I calmed the situation down by opening a dialogue specifically about the project and...We spoke about it for a while and he eventually calmed down, apologised and admitted he’d been under a...wherever it makes sense to start and try to be as chronological as possible — if you find yourself jumping backwards.... Your story should be too. Use drama Use any rhetorical technique you have in your arsenal to make sure...start point and it needs a good end point. One thematically pleasing option is to end at the present — ‘...

...algorithms are trained on vast amounts of data to calculate the relative immunogenicity score (Mattei et.al... evaluation of ‌therapeutic immunogenicity is typically performed using traditional 2D assays with immune...inform decisions about in vivo application and clinical testing. This would pave the way for improved ...in Macaca fascicularis: From Manufacturing to Preclinical Studies. In J. Bluemel, S. Korte, E. Schenck,...F. Weinbauer (Eds.), The Nonhuman Primate in Nonclinical Drug Development and Safety Assessment (pp. 519...2016). Antidrug Antibody Formation in Oncology: Clinical Relevance and Challenges. The oncologist, 21(10...