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We narrowed to 727 results for: cat.1

Showing: 61 - 90 of 727 results

  1. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

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    Blog Post
    ...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...  Option #1: Affinity tag purification You tag YFP and use an affinity column for purification. After binding...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...  Light controlled protein purification with OptoNBs The first application for OptoNBs takes us in vitro...light-controlled affinity purification of unmodified proteins. Affinity purification methods typically require...

  2. Viral Vectors

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    Collection
    ...material to specific cells in an organism. Figure 1. Using viral plasmids to generate virus. A) Viral ...of certain genes that are required for viral replication. Thus, as a safety feature, viruses used in research...Instead, the genes that are required for viral replication are usually divided among many plasmids, such...

  3. CRISPR Plasmids - Plants

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    Collection
    ...Hyg Chen 86196 pYPQ141-ZmUbi-RZ-As Maize ubiquitin 1 none As Cpf1 Qi 91715 pKEE401 yes, cut S. pyogenes...Gene/Insert Selectable Marker PI Publication Base Edit Catalytically dead dCas9 fused to a cytidine deaminase...Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to a transcriptional...Insert Promoter Selectable Marker PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs ...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted...

  4. Viral Production

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    ...to determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images). Figure 2: AAV...typically prepared using endotoxin-free plasmid purification kits. Resources Browse lentiviral preps available...

  5. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...target site. Benefits of truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated...protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA...gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site). When...

  6. Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)

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    Blog Post
    ...activity in single cells using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...which have previously been characterized. See Box 1 in Kudo et al. for more details on the design of the...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled...nuclear export and import rates can influence KTR translocation. Work from the Covert lab suggests that these...

  7. Plasmids 101: Modular Cloning Applications and Kits

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    Blog Post
    ...Figure 1: A chart with MoClo kit recommendations for different modal organisms and applications.  ...Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American Chemical Society (ACS), 4 Nov. 2015, pp...., vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021...Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. ...Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. ...Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://...Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July ...

  8. Pooled Library Amplifications

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    Blog Post
    .... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples...    Figure 1: Factors to consider when creating a pooled library amplification protocol. Created...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...ask for an amplification protocol with your deposit. This is because repeated amplifications of pooled ... an amplification protocol developed for each individual pooled library; generalized amplification protocols...Plasmids Variable Region Size DNA per Amplification Amplification Type Link to Protocol CRISPR Knockout... this blog, we’ll discuss how to develop an amplification protocol for your pooled library deposit. When...

  9. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    Blog Post
    ...in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...the deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA...

  10. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates the three... quantity.   Figure 1: Plasmid preps fall into different categories based on their relative qualities...volume ranges of culture as their input (Table 1). When you feel like you need just a bit higher DNA yield...kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume can ...beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount...Amount of culture Approximate DNA yield** Miniprep 1–5 mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep...column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications tend to...

  11. PCR Applications for Copy Number Variation Assays

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    Blog Post
    ...the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...run in triplicate.  The nice thing about qPCR is that since it measures relative quantification - that... not need to be run in triplicates. ddPCR is an extremely accurate application, even in areas of the genome...Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications Resources on Addgene.org...

  12. DNA Purification Without a Kit

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    Blog Post
    ...series to increase binding capacity. Figure 1: Purify DNA using glass filters. Image: Kim and Morrison...a gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method... solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2) it helps...property and developed a protocol that uses Whatman No. 1 paper or even paper towels to purify DNA and RNA from... DNA purification without a kit as well as one way to reuse silica columns from DNA purification kits....consider some ways to purify DNA without a kit. DNA purification kits have advantages: they are convenient and...because you’ve run out of columns. Kit-less DNA purification methods avoid many of the drawbacks of kits ...

  13. New Tools Enable CRISPRa for Neuroscience Applications

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    Blog Post
    ...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...genetic underpinnings of these processes has been complicated by the lack of precise genetic tools for modulating...recent advancements and offer commentary on their application to neuroscience research. What is CRISPR-based...accessible to neuroscientists, not all neuroscience applications require such optimized tools. This is elegantly...

  14. Neurodegeneration Research Collection

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    Collection
    ...one of three different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of...loci seem to be associated with the disease and implicate a wide range of cellular processes. The vast majority... consists mainly of managing symptoms through medication and surgery. PD primarily involves the malfunction...available from Addgene, sortable by type of genetic modification and target species. You can also find our guide...Sun et al. Nat Methods. 2020 Oct 21. Glutamate indicators with improved activation kinetics and localization...A multicolor atlas for whole-brain neuronal identification in C. elegans. Yemini et al. Cell. 2021 Jan...window) A news website and information resource dedicated to helping researchers accelerate discovery and...

  15. Early Career Researcher Toolbox: Social Media for Scientists

    Type
    Blog Post
    ... writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media as a scientist....needed to start using Twitter. Sharing Chemistry Cat memes use to be how I used social media as a scientist...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ...remotely as an Addgene Science Communication Intern. Developing science communication skills, while also learning...

  16. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...such a therapy, CRISPR/Cas9 would need to 1. excise the HIV-1 genome from every infected cell and 2. prevent...

  17. New Norepinephrine Indicators: nLightG and nLightR

    Type
    Blog Post
    ...norepinephrine indicators: nLightG and nLightR (Kagiampaki et al., 2023).     Figure 1: Properties...Article 6. https://doi.org/10.1038/s41592-022-01488-1 More resources on the Addgene blog Fluorescent proteins...after application of NE (10 μM) and corresponding pixel-wise ΔF/F0 heatmaps. White insets indicate surface...surface expression of the indicators over white dashed lines. Scale bars, 10 μm (HEK293T), 20 μm (neurons...trialed in two-photon in vivo experiments. nLight indicators are more sensitive to and selective for norepinephrine...They can, however, be studied with the nLight indicators. Speed One of the major advantages of this next...… Patriarchi, T. (2023). Sensitive multicolor indicators for monitoring norepinephrine in vivo. Nature...

  18. CRISPR References and Information

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    ...CMV-EGFP PDF, 2.4 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system...Labs. ACEofBASEs (Link opens in a new window) Identification of sgRNA target sites and off-target evaluation...number of potential off-targets, target's genomic location, and genome annotation. Available genomes are ...sequence, which can be an accession number, genomic location, pasted nucleotide sequence, or a sequence text... Target Finder (Link opens in a new window) Identification of gRNA target sequences from an input sequence...window) A collection of protocols, materials, and publications by members of the Division of Signaling and ...Download protocol Addgene CRISPR pooled library amplification CRISPR pooled libraries Protocol at Addgene ...

  19. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...wondering what her cat is thinking. Find her on Twitter @JessicaSacher.   References 1. Nobrega, Franklin...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...RBPs successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This...first step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that ...in the bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to...proteins expressed by your library. After blocking for 1 h at room temperature with gentle shaking, gently ...

  20. COVID-19 Resources

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    Collection
    ...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV... CD147-spike protein . Cathepsin L - Bosch BJ, et al, 2008, J Virol. Cathepsin L Functionally Cleaves ... is involved in virus entry into cells. ( 4 ) Cathepsin L - a cysteine endosomal protease that triggers...Andrew Ellington's lab. High-surety isothermal amplification and detection of SARS-CoV-2, including with ...

  21. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

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    Blog Post
    ...first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary electrophoresis...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...see any signs of DNA in your final tube after purification. How do you know if you actually have DNA in...you choose could be based on your downstream application, time, and instrument availability. UV absorbance... at 230 nm. The A260/A280 ratio is used as an indicator of DNA purity. Ideally, this number should be ...like a PCR product). This method also gives an indicator of DNA or RNA contamination based on the presence... about quantifying DNA In choosing your DNA quantification method there’s many things to consider: cost...

  22. Scientific Peer-review: Providing Critical and Kind Feedback and Advocating for Open Science

    Type
    Blog Post
    ...the journal-specific guidelines.  Peer-review step 1: The first read At the first read of the paper, I ...group of Prof. Mark Tester, where she focuses on (1) salt-induced changes in root-to-shoot ratio in Arabidopsis... to help decide whether work is suitable for publication in their journal. The early peer-review attempts...information that would prevent the study to be replicated or omission from the literature. I think that...Addgene.org Learn about depositing plasmids pre-publication Find molecular biology protocols Find eBooks ...

  23. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    ... Antigen-1 (oriP-EBNA-1) system where each vector contains a viral origin of DNA replication, OriP and...pCXWB-EBNA-1 is used in the reprogramming cocktail. pCXWB-EBNA-1 lacks oriP and is unable to replicate in human...and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences are necessary...expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...pCXLE vectors listed below in Table 1 are available at Addgene. Table 1: List of pCXLE episomal plasmids,...sufficient for retention and replication of the vectors in human cells [8]. The EBNA-1 protein recognizes OriP...obtained and the subsequent reprogramming success [1]. Fibroblasts need to be cultured and expanded in ...

  24. Split Fluorescent Proteins for Studying Protein-Protein Interactions

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    Blog Post
    ...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles...

  25. Antibody Guide

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    ...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...Handling Visualization Signal Amplification Application Overview Quantification Methods Capture Methods Visualization...Antibody Applications Antibody-based applications can be generally classed into four categories: Quantification... Read on to learn more about the applications common to each category, including an outline of the basic...these applications, visit the Antibody section of the Addgene Protocols page. Antibody Applications - Quantification...concentrates in the sample. Controls for quantification methods Quantification methods should be run with: a positive...

  26. Viral Vectors 101: Viral Applications

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    Blog Post
    ...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...their use in other clinical applications. In the last decade, viral applications in cancer treatment and ...s unpack the myriad number of emerging viral applications and give you some recommendations for putting...putting viruses to work. Viral applications Viruses work by infecting a host cell (the target of viral infection...expressed. It’s important to note that for all the applications we discuss, there is more than one type of virus...retrovirus, AAV, etc.). When you’ve decided on an application, our Viruses as Biological Tools blog can help...

  27. Retrograde AAV viral preps

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    ...NLS-mRuby2 Control Gradinaru 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Control Fishell 83895 pAAV-hDlx-Flex-GFP-Fishell...the functional catagories listed below. Narrow down the items available within a category by using the ...experiments can be found on the material pages for viral catalog items. Browse Available Retrograde AAV See our ... Tools Clear Filters ID Name Promoter Activity Category PI 37825 AAV-CAG-GFP CAG GFP Control Boyden 51502...offers you even more options. Select from a vast catalog of eligible AAV plasmids and we’ll make the viral...

  28. Transferable Skills Guide: Cross-team Communication

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    Blog Post
    ...Addgene, we use seven different types of “plates” (Fig. 1). When you have to tell an engineer that you want ...”? We took steps towards solving this problem by 1) making sure we qualified these words when we discussed.... I needed to a) figure out the sources of miscommunication and b) find practical solutions.  My PhD ... for this task. Good scientists learn how to communicate their work differently based on their audience... family is going to vary widely. The way you communicate your science at a poster is going to vary depending...two main problems with Addgene’s cross team communication. Although these real world examples did not ... for the container to be used in cross-team communication A list of “AKA” names, or jargon terms, for ...

  29. Great Results Start with Great Standard Curves

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    Blog Post
    ...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there is no linear relationship...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve...

  30. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...pooled library amplification. F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 gal-thi-1 supE44 λ-relA1 ...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...
Showing: 61 - 90 of 727 results