We narrowed to 842 results for: Eras

... In each case, we leveraged the team’s knowledge of the scientific domain in general and of our data in... that we needed to improve more than that: the general “findability” of the information on the site. Different...approached the issue from multiple angles: the overall user experience of our website, the different website...resources and handle millions of queries. In an iterative process driven by our user experience experts,...approach much more than if we were dealing with general purpose information. Below are a few of the strategies...

...interest, as it is prone to generating indel errors. Indel errors generated in the course of repair by ...vast toolbox of processing factors, including polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific...illustrated below, NHEJ-mediated repair of Cas9-generated breaks is useful if the intent is to make a knockout...overhangs is available. Cas9 can also be altered to generate a targeted single strand break; when two such ...Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular...

...a high imidazole buffer that outcompetes the interaction between the protein of interest and the metal...polyhistidine tagged protein of interest (purple) and several contaminating proteins (blue, green, orange) flows... low imidazole wash buffer disrupts the weak interaction of the contaminating protein but not that of ...A high imidazole elution buffer disrupts the interaction of the protein of interest and it is successfully...protein conformation, the polyhistidine-metal ion interaction is not disrupted by denaturants (Hochuli, 1988... of a protein. Neither the N or C terminus is preferable over the other however, tag placement can affect...

...PubMed PMID:29483642. Making protein-protein interaction studies easier with a 2in1 vector cloning system...cloning system is well suited for protein-protein interaction (PPI) studies in plants that rely on transient...Grefen Lab has utilized the 2in1 cloning system to generate a series of plasmids to study PPI using Förster...Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different, binary 2in1 vectors with all...powerful approach to control protein-protein interactions in cells because the protein binders can be ... that these photo-reversible protein-protein interactions can control subcellular localization processes...

... moment that Teytelman realized that running interactive workshops to train researchers on tools and best...next steps. April Clyburne-Sherin had already run several reproducibility workshops as Director of Scientific...to this open science collaboration. The first iteration of these workshops took place at Caltech in Jan...being adopted by a wider group of researchers. Generating material to empower others to practice and teach...team was assembled when it was realized that interactive workshops that train and provide researchers ...on the Addgene blog: Check out this guide on Transferable Skills: External Collaborations Learn about ...

...way they are, the research antibodies in your refrigerator often need a little help to be useful. Mainly...enzymes. The most common enzyme conjugates are Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP...This chemical coupling can be achieved through several different mechanisms. Most take advantage of chemical...sequence or that rely on strong protein-protein interactions with other peptides, like an Fc-binding domain...acids or peptide tags. 5) Strong protein-protein interactions with other peptides. Created with BioRender....Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues...

... only for translational research, such as gene therapy, but also basic research. To improve cell-type ... with high rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling...makes it easier to trace a neuron’s projections. Generating animal models: Transgenic animals allow for the...tissues, but they are costly and time-consuming to generate, especially for large animals such as primates...basic as well as translational research for gene therapy. The Roska lab hopes to design improved versions...blog Read more about using AAV for retinal gene therapy Find more about precise neuronal targeting using...

...interesting discoveries. You’ve analyzed the data and generated beautiful figures. And, you can’t wait to tell...need to first make your poster. While a poster generally contains the same sections as a primary research...references and contact information, and it is generally acceptable for the font here to be much smaller...is better! Your poster should be readable from several feet away. It can also be useful to use two different...it’s good to let your ingenuity shine, and you generally have free reign over how you put your poster together... as rows. It can be more catchy because it is generally less common and will stand out; however, be careful...

...and their use for HDR editing and mouse-model generation. Key components of Nanoblades Nanoblades are ...hematopoietic stem cells (HSCs) using Nanoblades. Overall, editing efficiencies were between ~50-70% for ...lower rates of off-target effects. Nanoblades generated up to 76% editing efficiency in mouse BM cells...-target effects, with electroporation of RNPs generally having lower off-target edits compared to DNA ...gRNA, Cas9, and a HDR donor template DNA can be generated by incubating Nanoblade particles with polybrene...Nanoblades are a potential alternative method for generating transgenic mice strains and for non-invasive ...

...prep is straightforward and rapid as there are several commercially available p24 ELISA kits that can ...plasmid was used in the transfection; if preparing several different lentiviruses in parallel, this approach...expansion, and confirmation. This procedure can take several months but should be considered in labs that will...virus or require precise titers. Additional considerations for titering lentivirus Volume and transduction...4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral vector...predicting gene transfer events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957. PubMed Central...

...them to see how feasible they were, conducting literature reviews and having weekly journal clubs outside...this process, we had an idea that survived the literature review, survived technical feasibility analysis...evenings on the project, but still continued to generate new ideas as they popped into our heads. Anything...color change until a few months later. RevBio accelerates with help from SynBio Axlr8r We limped along ...business. We ended up applying to a business accelerator freely stating that we had an idea, knew how ...rough day, and we have great partners in this accelerator program who want us to succeed. We’re excited...

...DNA, the CTD is also sometimes called the PAM-interacting (PI) domain. The guide RNA is bound primarily...most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, so it associates... less stringent recognition mechanism that can tolerate mismatches in this region. The crystal structure.... As mentioned above, however, Cas9 sometimes tolerates mismatches in the next 8-10 bases after perfect...this region of Cas9 also allows the protein to tolerate RNA:DNA mismatches between base pairs 18 and 20...Each new structural insight also spurs the next generation of engineered CRISPR enzymes with their own tradeoffs...Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization...

...experiments require some consideration of FP properties. It is important to look out for general problems associated... judgement by simply “looking at them”. Modern cameras and image analysis software are highly sensitive...microscopy cannot be interpreted as direct molecular interaction without further investigation. One fluorescent...microscopy method commonly employed for analyzing interactions between two molecules more closely is Förster...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

... copy number variation (CNV) assays. There are several varieties of these, but in this blog post, we're...length of the amplicon. As the probes can have several different markers, it can also be used to run multiplex...Do you need to call exact copy numbers or just general trends? How confident do you need to be in your...Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications...Applications Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer...SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination ...

...the conference After using your elevator pitch several times at the conference, you might notice some ...larger side? At large conferences, chances are that several talks will be going on at once in different rooms...a conference, be sure to practice beforehand. Generally, two types of people will visit your poster: those...have “tweep ups” where you can meet those you’ve interacted with on science Twitter in real life. If there...the tweep up (and for the entire conference, in general). After the conference Review your favorite conference... considered networking, but here are some more general tips. Before the conference Networking is key even...

...that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in human disease...and is stable and active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components...highly conserved glutamine-fructose-6-phosphate aminotransferase (glmS) gene in the recipient bacteria due ...Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease (S. aureus... library strains was measured after 6 and 12 generations with or without induction of CRISPRi knockdown...

... (cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate to dissolve and requires...carefully layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation... comparing to an AAV standard curve, which is generated from an ITR-containing plasmid. This whole process... always be included. While the physical titer generated by qPCR is useful, I test a few different concentrations...inverted terminal repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2....

...incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes ...could detect knock-in of tags at a target site. Overall, 64% of researchers were unhappy with the new method...the water. On the bright side, successful indel generation and epitope knockin was achieved by a few respondents...ssDNA leading to inefficient downstream editing. Overall, the real take-home message thus far, based on ...form a complex with a 5’-P-ssDNA guide at room temperature. Until that time, those who have been lucky with...Pooran Dewari is a postdoc at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. His research interests...

... Their focus at the lab is on plant-pathogen interactions, and her aim is to produce constructs and edit...biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens... share and educate and enable plant science in general. We are funded by a charity and since we are making...elements anymore. The kit contains sequences for several different fluorescent proteins. Visualizing fluorescence...piece of DNA. We do a lot of protein-protein interaction work and we’ve gotten some tools back from Addgene...Twitter account recently inspired by an academic in literature who gave a talk to the women’s research network...

...reporter enzyme. For multicolor staining, careful consideration must be used when selecting which antibodies...meticulously screening, optimizing, and characterizing several anti-mouse and -rabbit nanobodies. If you’re interested...described in Pleiner et al, the key steps for generating nanobodies are outlined below; no alpaca required...due to their N-terminal His14-bdNEDD8 tag. After several washes, this tag can be cleaved by the bdNEDP1 ...nanobodies from the column. bdNEDP1 protease can be generated using plasmid pDG02583. Label the Nanobodies: ...Maleimide-conjugated HRP can be found here and several maleimide-conjugated dyes are also commercially...

...infection; however this would not be great for a gene therapy vector! For viral vectors, only the protein for...viral vector particles are made while missing the polymerase gene may seem like a mystery. But it’s simple...transcription and translation. Here, we will discuss several aspects of the viral “life cycle” in order to understand...step 3), but note also that it is worthwhile to generalize the idea of “transduction” to mean any transfer...et al., 1973). Interestingly, retroviruses are generally only “pseudodiploid” in that — while they have... M., & Bishop, J. M. (1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis...

...incorporated a discovery-based lab on planarian regeneration into one of my own courses, I was excited to...phosphorylation on the enzyme’s structure, function, and interaction with other proteins.   It is important to...be authentic – students should be attempting to generate new knowledge that will be of genuine interest...and bench work to identify regions of MDH that interact with citrate synthase and presented their findings...increasing diversity and inclusion in STEM disciplines (Bangera & Brownell, 2014; Duboue et al., 2022), as they...classroom.   References and Resources References Bangera, G., Brownell, S. 2014. Course-based undergraduate...

... both vector amplification with low fidelity polymerases and the cost of synthesizing longer oligos, which...using the same three step protocol:  Fragment generation - Any linear DNA fragments can be assembled in... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...simple cloning procedures. Use high fidelity DNA polymerase This will minimize random errors during PCR: ...aliquots of PCR master-mix, containing buffer, polymerase and dNTPs, can be stored frozen: just defrost...mutagenesis by placing homologous ends on DNA using polymerase chain reaction." Biotechniques 10.1 (1991): 62...

... Major restaurant chains including Chipotle and Panera Bread have pledged to eliminate the use of antibiotics...number of phagemids would be needed for an in vivo therapy. Another strategy for CRISPR antimicrobials combines...replication-competent phages and antibiotics. Phage therapy fell out of fashion with the introduction of antibiotics...Rodolphe Barrangou. “A CRISPR design for next-generation antimicrobials.” Genome Biol. 15(11):516. (2014...Ido, Miriam Manor, Ruth Kiro, and Udi Qimron. “Temperate and lytic bacteriophages programmed to sensitize... phage therapy...

... to write, to read efficiently, to search the literature, and to synthesize a large volume of information...appreciate the discussion sections of the primary literature as a resource that both summarized results and... sure if I should cite reviews or the primary literature or both. I ended up citing both because I used...the scope and the content. Submit your article! Overall, writing a review can be overwhelming and challenging...Thompson, and Thomas D. Gilmore. "Histone acetyltransferases and histone deacetylases in B-and T-cell ...Addgene Blog Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Tips for Improving...

...ricin toxicity genes. The screen also discovered several genes previously not associated with ricin regulation...known ricin regulation genes compared to results generated using non-targeting guides as controls. While ...sites. Results from the screen also reproduced several previously observed characteristics that influence...activity: 1) mismatches closer to the PAM are less tolerated than mismatches more distal to the PAM, and 2)...off-target activity, Morgens et al drew some generalizable conclusions about off-target activity, demonstrate...Michael C., et al. "A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility...

...circuits and dynamics. FPs are generally bright, stable and well tolerated by cells. They can be used for...neither disturb POI folding nor its ability to interact with protein partners within the cell. However...can affect protein folding and protein-protein interactions. Also, overexpression of FPs can trigger protein...ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with facile ...monster” fluorescent proteins (smFPs). smFPs are literally composed of FPs fused to multiple epitope tags...

...was first developed in 2001, with successive generations developed ever since. GCaMP6 was offered through... added jGCaMP7 and eventually jGCaMP8, later generations of GCaMP6, to the service. While they were both...jGCaMP8 per year from 2016–2023. All three generations are regularly requested and a valuable part of...size of the incremental improvements between generations: jGCaMP8 allows for the “picture” of a train ... former approach and involves reading primary literature, soliciting information from requestors and depositors...Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...deliver all the protein and RNA components, and several sgRNAs must be screened to identify effective ones...Schematic of the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids used to express...gene expression. Gene isoforms refer to mRNAs generated from the same gene but with different untranslated...regulation — at least, more straightforward than generating cDNA for each gene isoform individually. To highlight...their genomic DNA sequenced. Guide RNAs targeting several known and some unknown regulators of neuronal differentiation...Although cDNA overexpression approaches may still be preferable for individual gene activation in select in vitro...

...enhancer sequence, then interact with mediator complexes and the RNA polymerase at the promoter region ...have allocated tremendous time and resources to generate highly specific tools to target different cell...allen-institute-aav-enhancers/","type":"EXTERNAL"},"user_generated_content":false}{% endraw %}{% end_module_attribute...sequences of DNA that regulate gene expression by interacting with proteins such as transcription factors and... A., Bickmore, W., Dean, A., Nobrega, M. A., & Bejerano, G. (2013). Enhancers: five essential questions...