...K855A) and eSpCas9(1.1), followed by a technique for detecting genome-wide double strand breaks (BLESS...it’s obvious that CRISPR is a ground-breaking technology that’s already had a huge impact on research ...effects on various biological disciplines, the base technology itself is constantly improving. Cas9 variants... "High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects." Nature (2016...

...developing assays.  Protocol architecture The basic (very basic!) architecture of the indirect approach is...direct approach isn’t giving you enough signal to detect your protein of interest, it may be time to look...occasion, usually after other signal amplification techniques have been ruled out.  Ease of access Signal amplification...course, this is much easier said than done! Each technique, and sometimes each individual assay, will need...

...recombination. What is Cre-lox? The Cre-lox system is a technology that can be used to induce site-specific recombination... lethality. To facilitate the use of Cre-lox technology, transgenic mice have been constructed that express...system is another site-directed recombination technology very conceptually similar to Cre-lox, with flippase...analogous to Cre and loxP, respectively. The FLP-FRT technology can be an effective alternative to Cre-lox, and...have any tips or tricks, or clever cre-dependent techniques?  Let us know in the comments! References 1....

...cell stage using one of several techniques. The longest standing technique is the injection of a targeting...before working with mouse models, several common techniques used to create engineered mouse models at embryonic...the pros and cons of different genome editing techniques. Inbred and outbred strains Mouse strains used...Cre is expressed.  Several variations of this technology can be used to customize the effects of this ...

...President's Council of Advisors on Science and Technology [PCAST] (2012). Engage to Excel: Producing One...Additional College Graduates with Degrees in Science, Technology, Engineering and Mathematics, Washington, DC:...DC: US Government Office of Science and Technology. 3. Duina, A. A., Miller, M.E., & Keeney, J.B. Budding...BioBuilder Use Addgene Videos to Learn about Basic Lab Techniques High School Students Explore Careers at Addgene...Molecular Biology Reference Pages Learn Simple Lab Techniques in Our Protocol Videos ...

...on to find out more about this amazing imaging technique and find further tips for using FRET in your experiments...proteins is not optimal, FRET may not occur or be detected even if the two proteins form a complex. To help...a potential source of error in suboptimal or undetectable FRET, well-characterized FRET reference standards...environment which alters the FRET efficiency. Detecting certain cellular changes with an intermolecular...the concentration or activity of small molecules, detect cellular processes and signaling cascades, quantify...

...science and technology park, it enjoys preferential governmental policies for a high-tech development ...development zone so many science and technology companies and start-ups choose to locate there. I made time to go...because they contain “hard stop” mutations. New techniques used by the Lowe lab have allowed them to begin... and Sun Yat-sen University presented two new technical developments. Using the Tet-on system, his lab...

...viral handling safety   Containment and personal protection are key to lab safety and the same principles...department if they are not currently provided. Eye protection or face shields are required whenever there’s...splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL... cabinet. Many researchers choose to wear eye protection when they are working in a biosafety cabinet,...organization. It’s always okay to add another level of protection! When centrifuging viruses (a necessary step...

...chromoproteins? One reason may be that chromoproteins protect corals from solar irradiation by reflecting visible...gives scientists the ability for instrument-free detection. Unlike fluorescence or luminescence, which require... fluorometers, or luminometers, chromoprotein detection can be done by the naked eye. This makes cloning...more time consuming and expensive methods. While techniques such as  blue-white screening require X-gal, ... (2000) Fluorescent pigments in corals are photoprotective. Nature 408:850–853 . https://doi.org/10.1038...

... use various techniques to control protein expression for experimental, biotechnological, and medical ...expression systems Plants provide a cheap and low-tech means of mass expression of recombinant proteins...desired protein. Despite being a rather nascent technology, plant cells have been used to express a wide...expression systems however, are expensive and very technically challenging to use. Alyssa D. Cecchetelli is...

... standard EGFP plasmid that used to be sold by Clontech and is in a freezer box in just about every lab...environmental changes that the biosensor is designed to detect resulting in false positives [2, 4]. Solutions ...molecular sizing columns and native gels fail to detect EGFP dimerization. Importantly, all of these assays...assays are performed in vitro. Assays to detect FP dimerization in in vivo cellular contexts, such as ... from nonbioluminescent Anthozoa species. Nat Biotechnol, 1999. 17(10): p. 969-73. PubMed PMID: 10504696...

...making them ideal for single-molecule fluorescence techniques. After generating a series of single- and double-tagged...be imaged using one- or two-photon microscopy to detect action potentials and subthreshold events, including...difficult with GEVIs. Not only was ASAP5 able to detect mEPSPs, it also enabled imaging of their propagation...protein and a NanoLuc® (Nluc) Luciferase-fused detector that specifically binds to active (GTP-bound) ...functional heterotrimers with endogenous Gβγ, enabling detection of activation triggered by endogenous GPCR. Example...

...around the globe. Our first story focuses on a technology that has changed virtually all of biology…CRISPR...repository. Other researchers, eager to try this new technology, were quick to request CRISPR plasmids and, in...favorite observations from this data is that CRISPR technology is consistently showing rapid development and...requested through the years, even as more CRISPR technologies become widely used and available. If we look...all of this work helped scientists use CRISPR technology? We think so — and so do our requestors! CRISPR...

...To achieve this, scientists use a multitude of techniques including co-transfection of two or more plasmids...started with them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially...straightforward process. Rather than try to directly detect your gene of interest, scientists have instead ...not have to exclusively be used as a means of detection; they are useful almost anytime you want to express...upstream of an IRES fluorescent reporter for easy detection.5  Just released: Addgene's Plasmids 101 eBook...

... last 5 years to incorporate more science and technology into the school curriculum. Our Parent Teacher...parents who can answer some of the surprisingly technical questions elementary students can ask. It doesn...and the internet before it, genome engineering technology is moving along much faster than it can be officially...sugarcoat the potential dangers of people using the technology in an unethical manner, but I also tried not ... timely.” Genome engineering, and many other technologies and fields in biology, are moving so quickly...

...between neighboring fluorophores, FRET can be used to detect the inter- and intramolecular interactions that... fluorescence complementation is a method for detecting protein-protein interactions that uses split fluorescent...FP results in a fluorescent signal that can be detected by fluorescence microscopy. Figure 1: How...Medical Center, where he used molecular biology techniques to study inflammation and neurodegeneration. ...BiFC): A 5-year update and future perspectives. Biotech 53. https://doi.org/10.2144/000113943 Additional...

...ELISAs. Immunofluorescence. What do all of these techniques have in common? They all typically require secondary.... Could there be an alternative method for immunodetection? Enter the Görlich lab and their anti-mouse...antibodies for all of the above listed molecular techniques, but they can also be expressed in bacteria and...main reasons antibodies are great indirect immunodetection tools is their strong signal strength. This...Although antibodies have been the titans of immunodetection, nanobodies may be better suited for the job...

...recombinant Erythropoietin. J Biotechnol. 2020 Feb 10;309:44-52. doi: 10.1016/j.jbiotec.2019.12.017. Epub 2019...conformational changes in the antigen or lower detection sensitivity of the assay. Where possible, it can...beads from very high cell density suspension. Biotechnol Bioeng. 2021 Sep;118(9):3499-3510. doi: 10.1002...Roberts CJ. Protein aggregation - Mechanisms, detection, and control. Int J Pharm. 2018 Oct 25;550(1-2...

...Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance companies, and governments...strategy tend to be mostly engaged by pharma and biotech clients. In this realm, there are a few different...organizations, investment and equity research firms, biotech start-ups, venture capital firms, and more. Essentially...science, with a newfound appreciation for how technologies get developed and commercialized after they ...healthcare industry publications like FiercePharma, FierceBiotech, and BioCentury can be an engaging way to learn...

...specific protein out of a complex mix. Using this technique, users can look for the presence or absence of...acids. Read on to learn more about this versatile technique.  Immunoprecipitation Overview An immunoprecipitation...species as the antibody that will be used for detection in a subsequent Western blot. When choosing beads...application. Typically, if the target protein will be detected in a Western blot then it can be eluted by boiling...epigenetics routinely use the chromatin IP (ChIP) technique to identify DNA binding proteins involved in histone...

...Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the absence of Cre. ... are determined using a chromogenic endotoxin detection assay based on the amebocyte lysate method. Purity.... mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from Karl Deisseroth (Addgene...for mycoplasma contamination using mycoplasma detection kits. Cell line is maintained for ~20 passages...

...RNA production can be detected by using a MS2 coat protein that specifically detects a specific RNA stem...instance, pleckstrin homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008...Cas9 is a protein, a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP...fluorescent proteins (Chernov et al., 2017) may improve detection. Since longer wavelengths have better tissue penetration...

...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef...protein-protein interactions in proximity labeling techniques such as BioID (Roux et al., 2012) and APEX (Rhee...tools, such as gene manipulation and mutagenesis techniques, are available to the scientific community.  ... as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent Advances on Physiology...

...Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide...naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR;...plasmid digestion (blue). Digestion with enzyme “B” detects primer multimerization (purple) which is evident...~3%). Note that primer duplication will evade detection in the initial RFLP analysis simply because the...on the Addgene Blog Brush up on Your Cloning Techniques Learn More about Plasmids in Our Plasmids 101...

... (reviewed in Shcherbakova et al., 2018). For detection of protein-protein interactions, we recommend ...Multiplexing and Optogenetics across Scales. Trends Biotechnol 36, 1230-1243 (2018). Shcherbakova, D.M. et al...Sivanesan, D. & Michnick, S.W. An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions...encoded calcium indicator for in vivo imaging. Nat Biotechnol 39, 368-377 (2021). Shcherbakova, D.M. Near-infrared... fluorescent protein for in vivo imaging. Nat Biotechnol 29, 757-761 (2011).   ...

...authors use PhoCl in combination with this caging technique to beautifully demonstrate light-dependent Gal4... base editors The popular CRISPR base editing technique uses dCas9/Cas9 nickase fused to a cytidine deaminase... PubMed PMID: 28585549 Kim Y, et al. Nature Biotechnology. 2017. PubMed PMID: 28191901 Listen to Our ...a fully sequenced genome, many applications in biotech, and is considered the most studied Gram-positive...vivo. Utilizing CRISPR interference (CRISPRi) technology, the arrayed knockdown library targets 289 genes...In Narayan et al., they used this now classic technique to elucidate the mechanism by which SOX2 acts ...

...understanding the brain’s function is knowing its architecture, in particular the connections between different...nervous system With the advent of viral vector technology, new methods have been developed to study neuronal...consistent which may be caused by the diversity of techniques, species, and brain regions used. Evidence for... Gradinaru Lab at the California Institute of Technology generated novel AAVs (Chan et al., 2017) that...lower toxicity, higher specificity and reduced technical variability compared to chemical tracers. rAAV2...targets neonatal neurons and adult astrocytes. Nat Biotechnol 27, 59–65. PubMed PMID: 19098898. PubMed Central...

...pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...activate the pathway than traditional pharmacology techniques allow. Multiple photoswitches can be used in ...reading Abreu, N., & Levitz, J. (2020). Optogenetic Techniques for Manipulating and Sensing G Protein-Coupled...References Abreu, N., & Levitz, J. (2020). Optogenetic Techniques for Manipulating and Sensing G Protein-Coupled...

...their hot-off-the-presses papers, the awesome new technology they’re utilizing, and the tricks they’ve used...plasmids in your own lab. Summaries of the latest technologies and breakthrough research. Insights about ..., post-doctoral fellow, lab manager, research technician, or Principal Investigator. How-to guides for...a special focus on cloning and plasmid-based technologies. Commentary about scientific sharing and the...

... Chapter 3 - Addgene as a leader in standardized tech transfer and the adoption of the UBMTA Chapter 4... and Diplomacy, with a focus on international technology management, from the Fletcher School at Tufts...investment banking analyst at Credit Suisse’s Technology Group in Palo Alto, CA.  Benjie Chen is a co-founder...and networks. Benjie was Addgene's Director of Technology for 7 years, and is now Chief Software Engineer...