...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells ...  After identification of ABE activity, XMAS-TREE can also be used to purify these modifications and work...ABE-targeted cells By Ellie Adams Single base modifications through ABE or CBE's have the potential ability...point mutations. They allow single-nucleotide modifications to edit DNA without requiring a double-stranded...mCherry and GFP expression to check for ABE’s. b) Quantification of base-editing efficiency using XMAS-TREE-...methods at various genomic loci in four different categories.     Brookhouser, N., et al. BMC Biol 18...MYC-dependent cancer cell lines. It can be applied for identification of essential MYC binding sites in a wide range...

...vesicles. Okada CY, Rechsteiner M. Cell. 1982 May;29(1):33-41. PubMed. Protein transduction: unrestricted...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...cassette is removed, the effector is expressed. By truncating one of the recombination sites in the construct...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...https://dx.doi.org/10.15698%2Fmic2020.04.713 Truncated TALE-fluorescent protein fusions as a DNA staining...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

...occur, but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...scientific and technical training is that these educational paths prepare you for a vast selection of career...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...relatively little tissue absorption, weak tissue scattering and small amounts of tissue auto-fluorescence...

... contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...extension. Now, they split EB1 into two halves (Fig. 1): one containing the microtubule binding domain and...cells, and has potential use for a wide range of applications in cellular biology. Van Haren J et al. Nature...versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity ...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to... The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry fluorescence is...frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply involves tracking...

...variants. This resulted in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the...lymphocytes, too, making them a versatile tool for application in both neuroscience and immunology. Don’t be...imaging for beginners Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans,...give it a shot. “Beginners sometimes think it’s complicated, but actually it is very good,” he said. “It ...his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing...

...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...ll be talking about the ins and outs of this classification system, including its history, biology, and...repeatable phenomenon, Gram began using it as a classification system. Eventually, any bacteria that retained...permeable to different classes of antibiotics. The classification system is also useful in a laboratory setting...bacteria type. The gram positive and negative classification system is incredibly helpful in visualizing...stepping into a lab.   Anna Kamens is a science communication summer intern at Addgene and a rising third-...

... cell types and physiological states. References 1. Ingolia, Nicholas T., et al. “Genome-wide analysis... While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second. They also explored...Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent...

...mature HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central ...antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based...

...Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed. Find the Plasmids @Addgene... interference (CRISPRi) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease...library of characterized aptamers and supporting publication: http://www.freebase.com/base/aptamer Cruz-...

...heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the enhancer region of ...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...light-activated-dynamic-looping (LADL) system. The group fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they...

...process is explained more fully in the animated Fig. 1 below. Normalization is a fundamental and often understated...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known...of Toronto. He is an author of 20+ scientific publications and patents.   ...

...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic.... Using multiplexed gRNAs can target multiple locations in the genome to modify, whether it be editing... use is pX333 from the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two ...thus marking cells that have undergone genomic modification. In this system, Cas9 must be supplied on a ...with Cpf1 Additional Resources on Addgene.org Catch Up on Your CRISPR Background with Our Guide Pages...

...present in the data such as synapsin, hsyn synapsin-1, human synapsin, hSynap. Matching We capture multiple...that allows category-specific, narrow searches. We defined three top level categories — catalog, educational... in a given category, and serve as a shortcut to a search scoped to that category. Catalog ID suggestions...prefer to browse and discover items in our catalog or educational resources through our main dropdown menu... of Addgene’s customer’s needs. Categories We created a categorization of our information in order to ...depositors — each with subcategories such as plasmids and viral preps for catalog, blog posts and protocols...80,000+ reagents and 700+ educational resources — including blog posts, protocols, guides, and collections...

...red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system is spread...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can... also be used to engineer new pathways with sophisticated gene expression controls. Most cell types do...light; these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...; only two plasmids are required per system. Applications of these systems The large dynamic range of ...suitable for various scientific or engineering applications.   References Refactoring and optimization ...Oct 11. PubMed. Find the plasmids from this publication at Addgene. Synthetic biology: engineering Escherichia...

...instead conveniently provide them on your smart phone. 1. Lab Archives by LabArchives The single, most important...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information...

...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...compiled a list of three options from the Addgene catalog. These were a miR30 cassette, Syntaxin3, and osa-miR528...

...to do that than in the Addgene blog?  Fig. 1: One of our favorite content collections is our Help...Our educational content  Have you ever wondered how we choose topics for the Addgene blog? Or why we...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...antibody and content team, we identified what applications should be included and what basic information... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise...through an editing process and final check before publication.   Do research! Nearly everything we write entails...Guide figures. This requires a fair amount of communication between our content team and our graphic designer...