We narrowed to 718 results for: des.1

...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process...Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources..., the use of LIC is often limited to specially designed plasmids. Sequence- and ligation-independent cloning...the “damage” and producing a complete plasmid. Besides being compatible with any vector, the SLIC protocol...

...part of) figure 1, uploading it as a preprint, or documenting it in some other way. Besides the data, new...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...Amsterdam. The previous two posts in this series described a practical approach to selecting a bright fluorescent...

...a glossary at the end of this post! Figure 1: Base transitions are edits between the purine bases...uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467-024-49343-5...uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467-024-50012-w...an abasic site. Like in most other base editor designs, the Cas9 nicks the non-edited strand to encourage...

...offers insight into a career as a Professor. Step 1: Your PI forwards you a Request For Applications (...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and...application. It typically includes overview information, key dates, funding description, research plan, award... setbacks, and potential alternative routes, Provides opportunities to convince others why a project...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...literature and offer educated insight into what the desirable features of future technologies should offer in...

...of interest and the metal ions.      Fig. 1: A sample containing a polyhistidine tagged protein... Bio/Technology. 1988; 6: 1321–1325. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg... for nonspecific binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins... I place the tag and how many do I need? When designing a plasmid containing a polyhistidine tag, you’...

...in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene activation...therapies to clinical trials in 2021. Figure 1: Schematic of the human MECP2 locus and MECP2 protein...Tillotson R, Selfridge J, Koerner MV, Gadalla KKE, Guy J, De Sousa D, Hector RD, Cobb SR, Bird A (2017) Radically...that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides during neuronal maturation (Tillotson...If you’re new to Rett Syndrome, this blog post describes the history of Rett Syndrome research, the role...MECP2 in Rett Syndrome Rett Syndrome was first described in 1966 by Austrian physician, Andreas Rett, who... his observations in a German medical journal. Decades later and after years of painstaking work trying...

...on a transport inhibitor response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal...and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin...repository that will help epigenetic researchers custom design their own engineering plasmid tools in about a ...cloning, the Haynes lab’s Protocols.io protocol describes an elegant system that allows for the assembly... cells that make up the human body and G-baToN provides versibility in tracking and modulating the interactions...interests via CRISPR techniques. This tool-kit includes an array of FPs as well as additional useful tags...libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent motif (PAM...

...halorhodopsins to silence neuronal activity (Figure 1) (Wiegert et al., 2017). How to get an opsin into... rather than aversive stimuli. In a recent paper, de Jong and colleagues used rAAV FLEx-based vectors ...brain regions associated with motivated behaviors (de Jong et al., 2019). FLEx vectors represent, therefore...developmental biology at the École Normale Supérieure de Lyon. Her love for educational science led her to...neuroscience 8.9 (2005): 1263. PubMed PMID: 16116447. de Jong, Johannes W., et al. "A neural circuit mechanism...scientific communication manager at genOway. A few decades ago, the brain remained elusive, not from a lack...Boyden et al., 2005; Deisseroth et al., 2006). Despite its relatively recent development, the history ...

...all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of...is available as a kit from Addgene.   References 1. Chen, Baohui, et al. “Dynamic Imaging of Genomic ...ortholog requires a different PAM sequence, a gRNA designed for one dCas9 should be specific to that ortholog...boxB Magenta boxB-MS2 White boxB-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently...genome editing. Such a system has been previously described for transcriptional activation/repression and ...

...state of a system that, despite all of its successes, has many deep-rooted flaws (1). As we discovered through... Follow David on Twitter @driglar.     References 1. Alberts, Bruce, et al. "Rescuing US biomedical research...all bad, however - there are some signs that the tides may be turning. In 2013, the NIH announced its first...in Scientific Training (BEST) awards, which are designed to support new approaches that address the need...

...the lab running like a well-oiled machine. Lab tip #1: Glove trick Putting on lab gloves can sometimes be...distribution. Lab tip #3: Barcode everything Use barcodes and scanners to keep track of reagents in your...materials quickly and easily. Learn more about using barcodes. Check out our “Lab Tip” filter on the blog...

...bioengineering, but it wasn’t clear whether they could 1) be decorated with proteins and remain functional,...Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. ...of R bodies R bodies have been studied for many decades, and they were known to be resistant to detergents...

...available in your microscope setup. Figure 1: Excitation (dotted) and emission (solid) spectra ...spanning nearly the entire visible spectrum (Figure 1). The GCaMP series (e.g., jGCaMP8) remains the gold... from University of Calgary. Over the past few decades, genetically encoded calcium indicators (GECIs)...

...The main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity.... targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins and genome editing...in one direction. Cas3 is thus not helpful for designer genome edits, but it is effective at wiping out...David, Chen, S., Janice, et. al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...method seems simple, I found it hard to remember to describe all four parts of a STAR answer once the interview... because sometimes companies have relaxed dress codes for interviews. You can also ask HR for clarification...

...impartial. The best science happens when scientists have 1) the courage to make mistakes and 2) the integrity...policies that “muzzle” government scientists to the de-funding of crucial environmental research programs...of what I saw are that we, as a society, need to: 1) use more citations for out data so our sources are...and minority communities, where these issues hit hardest. According to the Guttmacher Institute, most women...was supremely proud of Nashville that morning. Despite the rain, about 4,000 supporters of science showed... an agenda that halts current data collection, destroys existing data, and guts future funding.I marched...science belongs to the taxpayers who fund it, and we deserve to know the truth about our world. Jonathan Aguilar...

...for locating and containing escaped flies. Nature 4:1-22. https://doi.org/4.01.2022/hafd... research, particularly genetics research, for decades. Its short life cycle, reasonably close genetic...

...scavenger hunt and the subsequent clue. (Begin with clue 1 below!) You will find five photos this way. At the...and have unleashed them all over our recently redesigned blog. 🐕 Your task is to find them, scavenger...

...basis of many molecular biology tools! Figure 1: Common functions of natural plasmids. Created with....2024.01.039 Garcillán-Barcia, M. P., Redondo-Salvo, S., & de la Cruz, F. (2023). Plasmid classifications. Plasmid...difference is virulence plasmids! This category includes genes that help bacteria invade hosts, including...resistance to toxic substances like heavy metals or pesticides, enabling their hosts to survive where others...found on an E. coli fertility plasmid that also encodes a toxin, CcdB, and the resistance to that toxin...

...technologies (Bradbury & Plückthun, 2015a).   Figure 1: (Top) Polyclonal antibodies are produced in animals...Rahman MZ, He L, Xin L, Shan B, Li M (2016) Complete De Novo Assembly of Monoclonal Antibody Sequences. Sci...antibody function, improve antibody stability, and design tools that we haven’t even imagined yet. References...

...Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical...that actually bind antigens.      Figure 1: IgG antibody structure with important features labeled...talking about here? Many of the Fab fragments described above can be used for similar applications and...

...interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface ...populations of cells in a complex tissue or sample (1). When combined with cell sorters, this technology...assessed for the desired parameter. A droplet containing a cell that is positive for a desired parameter is...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...degree of scatter is captured by detectors and provides useful information about the cell. Light that ... traveling is called forward scatter (FSC) and provides information about the relative size of the cell...initially traveling is called side scatter (SSC) and provides information about the cell’s complexity. A cell...

...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0...2.0–2.2 For the mildest stripping solution, omit the Tween 20. Once your solution is made, you’ll...

...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small ...tag size, which can also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V...degradation of the tagged protein. Degrons are destabilizing domains which will be identified by various ...proteasome for degradation. Like several other tags described here, the dTAG uses a small molecule, a tag, and...protein for imaging. Given its popularity and widespread usage, a proteolysis-targeting chimera (PROTAC... The auxin-inducible degron (AID) system is the oldest system reviewed here. The AID system relies on ...Thankfully, this is a hot topic with many resources! Bondeson et al. (2022) reviews all the tags discussed here...

...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet...fluorescent output. The GEVI family of gadgets includes Ace, JEDI, and many more. They can be targeted...component as well as a small molecule which we will describe in more detail below. Types of Voltage Indicators...Drosophila to phosphatases from sea squirts. This design structurally separates the two components of the...membrane, a concern with several of the previously described systems. Several adaptations of this method have...voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or both. Targeting these systems...

...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...append their proteins of interest with unrelated peptides that already had high quality antibodies available...existence of high quality antibodies for well defined peptides. Munro and Pelham’s first published peptide tagging... tumorigenesis.  Using two different synthetic peptides corresponding to linear sequences within the middle...that perfect. One of the known limitations was described by Schüchner, et al., who published a paper showing...works and allow users to better troubleshoot and design experiments. It turns out that even with the most...

...editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes...dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the...tools, they do have undesired off-target editing effects. ABEs have been described to introduce base changes...efficient. Informed by these mutations and previously described TadA mutations (Gaudelli et al., 2017), they modified...heterodimeric fusion of wild-type TadA and TadA7.10, they designed one set of ABE8’s similarly (ABE8.x-d). In order...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning...end-joining, but will also efficiently ligate many undesirable structures, including substrates containing gaps...fidelity") better than T4 DNA Ligase (21, 25, 26). Despite this higher fidelity, Taq DNA Ligase can still ...possible NN pairs in the template. The probes were designed such that each possible pairing resulted in a ... high fidelity ligase, along with careful probe design and reaction condition optimization, will be critical...

...fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins and their ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence... in the wildtype protein and facilitating its widespread use in research. Many other mutations have since...array of GFP-based fluorescent biosensors has been designed to detect a variety of intracellular conditions...

... necessary for advancing the field. References: 1. Internal initiation of translation of eukaryotic ... of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim et al...adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in ...plasmids) are designed to have one or more of the genes replaced.  Additionally, 2A peptides could be PCR-cloned...PDF that you can save on your desktop. The Plasmids 101 eBook is designed to educate all levels of scientists...incorporated into plasmids for research purposes are described below. IRES Elements Translation in eukaryotes...the specific genes cloned into the vector.3  2A Peptides To overcome some of the disadvantages of the IRES...

...true reproducibility. The following are critical: (1) connectivity to other data sources used in research...engagement with the Wisconsin research community were to: (1) prevent vendor lock in by making it easy to get data...reproducibility than others. Unfortunately, most ELNs are designed as closed ecosystems and act as data silos because...RSpace ELN supports reproducibility RSpace was designed in response to requirements that emerged from ...used in research, and data repositories. Below I describe how each of these kinds of connectivity facilitates... in institutional and lab servers specifically designed to hold large amounts of data. Although it may... use of other tools they use in research. This includes chat apps like Slack, Microsoft Teams, and Google...

...be selected for these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed PMID: 22368488. PubMed Central PMCID...Researchers have used oligonucleotides in fluorescent visualization techniques for decades, most notably for... exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a ...aptamers as well, and over the course of the next two decades, these nucleotide-based ligand binders would prove...their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells (even...counterparts, antibodies and enzymes, such as: Design and selection for targets can be done largely in...

...type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467... depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Delivery to Intended REcipient Cells Through Envelope Design). This modular approach combines an interchangeable...LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes...that had been trafficked from the “source” to “destination”. Images adapted from Lee and Cheah et al. 2023...Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4 protein ticker tape recording. B) HEK ...

...membrane targetin sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. ...Reports. 2020. https://doi.org/10.1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few...their BioRxiv post, the authors generated and described several improved genetically encoded fluorescent...Takayuki Iwaki, Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion...contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag...expression can vary significantly depending on transgene design and delivery. To overcome these variations in human...Addgene The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting of CRISPR/Cas nucleases...

...writing and hopefully increase your success rate. 1. Submit your grant early! First off, and perhaps the...involved so it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback...to a lay audience. My lay paragraph typically includes a couple of sentences of introduction to my area...

... expanded to include even more loci. Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 ...researchers at Japan’s Hiroshima University who described their new tool in Scientific Reports in June. ... gap. As Sakuma and colleague Takashi Yamamoto describe in their recent study, the construction system...vector-mediated multiplex genome engineering. The system now provides an efficient targeting strategy for multiplex ... validated and ready. “Currently, the kit only includes two versions of Cas9, nuclease and nickase, but...Addgene here. Other Popular CRISPR Posts: How to Design Your gRNA for CRISPR Genome Editing CRISPR-Cas9...

... to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example, ... communication, and public health.     References 1. O'Neill, J. "Tackling drug-resistant infections globally... defined as microbial survival despite exposure to antibiotics designed to kill them or to impede their...that the term ‘antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because...different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria that...equipment, etc. Virtually any preventive measure designed to help keep you from getting sick can be helpful...

...here to listen to the interview with Tom McFadden!   1. Rosalind Franklin vs. Watson & Crick - Science History...One of Tom's earliest and most popular videos. Describes fossil rocks and their importance in understanding...

...editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted... JA, Han Y-C, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, Lowe SW, Ventura A (2014) In vivo engineering...targeted organs or tissues with Cas9 and a gRNA designed to target a mutated gene. In a Scientific Reports...Center are now available at Addgene along with a description of their experimental protocol. The three plasmids...plasmid tools, find more plasmids, CRISPR technology guides, FAQs, and CRISPR resources on Addgene's CRISPR...Additional resources on the Addgene blog How to Design Your gRNA for CRISPR Genome Editing by John Doench...

...glorious trophy. Sure it’s plastic and you have to share 1 trophy among 6 people, but you’ve earned bragging ...occurring earlier but that the photo evidence was destroyed to protect the innocent revelers. Our Halloween...for Halloween and how seriously we take costume design. In case you need more convincing, I’ve got just...to great customer support by dressing up as our Zendesk email system.  Our 2013 winners dressed up as ...

...does not greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function... conditions FLAG DYKDDDD or DYKDDDDK or DYKDDDK 1 Affinity and Purification Good for antibody-based ... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...depending on their protein of interest. After several decades of trying to address these challenges, researchers...examples. The tables list more common tags than are described in the post, but have been categorized to help...

... cerevisiae (Tuller et. al., 2010). Figure 1: An overview of peptide synthesis. A ribosome interacts...McClain WH (2000) The G·U wobble base pair. EMBO Rep 1:18–23 . https://doi.org/10.1093/embo-reports/kvd001...Usage Tabulated from GenBank database (CUTG), that provides an electronic dataset for codon-usage of 257,468...(A) The codon wheel depicts the 64 codons that encodes the 20 amino acids and three stop codons. (B) Example...Codon Optimization  ATUM DNA2.0  IDT Synthetic gene design made easy GenScript- GenSmart Codon Optimization...

...base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with...follow-up posts: (1) (2). Base editors have a narrow editing window of only a few nucleotides, so they are ...or one pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine...engineering and protein delivery. Nature Communications, 8(1), 15790. https://doi.org/10.1038/ncomms15790 Richter...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s...named ABEmax. In 2020, two papers were published describing additional ABEs evolved from ABE7.10 that have...

...alternative detection methods.      Figure 1: The four types of ELISA are direct, indirect, sandwich...conjugated antibody is not readily available. Table 1: Overview of the advantages and disadvantages of each...staining. Antigen-containing samples often include undesirable molecules that may bind non-specifically to assay...specific than other methods. In addition, the assay includes both primary and secondary incubation steps, increasing...

...Spaghetti Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins... expanded use of smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes...map these neuronal networks. For almost three decades, recombinant Adeno-associated virus (AAV) has been...symbol of opposition to the teaching of intelligent design in public schools and was started in 2005 in Kansas...

...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity .../35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic...

... present in the sample (APC in the case of Figure 1). In Figure 1A, the population needs to be shifted... decrease the compensation value.   Figure 1: Compensation example with single color control. Flow...This will make it difficult to properly gate your desired cell population. By compensating between fluorescent...

...for example, split cells on a Monday diluting them 1:10, you should be able to split on the following Thursday...Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis...and could be contaminated.Learn how Addgene uses barcodes to track samples Keep track of passage numbers...

...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs have ...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic... al., BMC Molecular Biology. 2013 August 20th; 14(1). TRICK: A method for visualizing the first round...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...by insertion and deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files...keep your product to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with...screening by PCR may be the way to go. Design a PCR to flank your desired edit site and run the PCR product...nucleotide along with your desired edit. This silent mutation should be designed solely for restriction enzyme... updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells... your cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...