...of human codon-optimized  cryptochrome 2 (CRY2) from Arabidopsis thaliana. When exposed to blue light, ...protein. After fusing the CRY2 and CIB1 domains to separate proteins of interest, a researcher can cause them...

...dimerization-dependent protein interactions. FEBS letters, 591 2, 415-424. PubMed PMID: 28032891. This is a different...feature is particularly useful for in-depth characterization of dynamic protein complexes. Split-BioID,...of the bait proteins’ interactome. Further characterization of these candidates is critical to understanding...

... already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step...present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene (e.g...model.  If your expression system is not well characterized in terms of CRISPR use, you will want to invest...

...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...vincristine, are regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our ...   C. roseus is not a model organism such as Arabidopsis or tobacco. Studying gene function in C. roseus...method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium infiltration into ...expression assay by agroinfiltration of leaves." Arabidopsis Protocols. Humana Press, 2006. 225-229. PubMed...transient gene expression analysis in seedlings of Arabidopsis and other plant species." Plant methods 5.1 (...expression method for analyzing gene function in Catharanthus roseus seedlings." Frontiers in plant science...

...system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to...experimental parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter is ...Photostability can be affected by experimental parameters (e.g. excitation light intensity, pH or temperature...

...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...QC'ing data at Addgene is like standing under a Niagara Falls sized rainbow   Over the years we have...pipelines, including bioinformatics. We loved the parallels between Seqera’s mission and our own, as both ...the sequential tasks in our pipeline, we wrote a separate Nextflow process. In Nextflow, a process defines...concept of channels and implemented the input declaration correctly, the script block in the second process...

...inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor expressed in the human...colleagues Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo and have used...

...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...transcriptome engineering References Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable...CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021...

...overview of these new variants:   Figure 2: Inner mitochondrial membranes imaged with structured...and screening that led to identification and characterization of AAV.CAP-Mac. Image reproduced from Chuapoco... Endothelial-specific AAV delivery By Cythina Arokiaraj (Gradinaru lab) Another useful viral tool deposited...

...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent...xCas9 3.6 displayed the second best PAM range. Characterizing xCas9 in mammalian cells Hu et al. compared...SpCas9. Hu et al. performed GUIDE-seq analysis to characterize genome-wide off-targets, since increasing PAM...

... Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed...few new ones that are not used so much but have comparable expression levels to things people do use a lot... developed mostly with model plants in mind, Arabidopsis and Nicotiana benthamiana. N. benthamiana is ...

...strain (which will be covered in more detail in part 2 of this blog series) controls the cell types in which...derived from random crosses between mice of uncharacterized backgrounds to create a limited but genetically...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking at...of the theme of my interview stories as the characteristic which the story demonstrates I possess.   Bullet...

...of these issues are not particularly new. Chapter 2, “The Professional Scientist,” addresses the challenges...over two decades, attempts to answer it in five paragraphs, with examples. If that sounds ambitious, that...them wanting to learn more. There are individual paragraphs that may have warranted their own chapters, and...

...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...or diluted as the cell grows resulting in the appearance of unmethylated sites in the chromosome. The ... without plasmid addiction systems. For example, Cara Morin and James Kaper used the hok/sok system to...223.1 (1992): 41-54. PubMed PMID: 1370544. Morin, Cara E., and James B. Kaper. "Use of stabilized luciferase-expressing...

...mrr-hsdRMS-mcrBC) Phi80lacZDeltaM15 Delta-lacX74 recA1 araDelta139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG ...+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ...mrr-hsdRMS-mcrBC) Phi80lacZM15 Delta-lacX74 recA1 araD139 Delta(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG ...Invitrogen F– mcrB mrr hsdS20 (rB–, mB–) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20 (StrR ) xyl-5 λ– leu ...indicated, the antibiotic powder can be dissolved in dH 2 0. Addgene recommends making 1000X stock solutions...million DNA fragments, which is far too many to separate from each other for the purposes of isolating ...recombinant DNA technology, which enables the preparation of more managable (i.e., smaller) DNA fragments...

...al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters...at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...to fulfill orders.  The barcoded tubes help to guarantee that no samples get lost during their journey....Addgene Get help with plasmid transformation, preparation, and analysis from our protocols and molecular...

...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...PMCID: PMC552224. 4. Gustafsson, Claes, Sridhar Govindarajan, and Jeremy Minshull. "Codon bias and heterologous...: 346-353. PubMed PMID: 15245907. 5. Maertens, Barbara, et al. "Gene optimization mechanisms: A multi‐...

...Henrietta Lacks. New York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University...line identity and purity making it easier for comparable studies to be performed on a cell line even decades...preferably some of these vials will be stored in a separate backup liquid nitrogen store to prevent loss from...

...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous...3645–3650 https://doi.org/10.1073/pnas.0909910107 Varani G, McClain WH (2000) The G·U wobble base pair. ...

...unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4...sequencing of neurons from each class to fully characterize the transcriptome. The first data release for...

...non-specific binding in our IHC condition.   Fig. 2: Schematic of the goldilocks antibody selection process...cross-reactivity (binds to non-target proteins), variability (separate batches can perform differently – happens when...

.... Find Plasmids from the publication at Addgene 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed...the project were able to move forward quickly in parallel. More broadly, we drew on the combined creativity...

...of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO...intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017) and levels of the second messenger... a “Top” and “Bottom” portion, each fused to a separate piece of RNA. Fluorescence is observed when these...23488798. PubMed Central PMCID: PMC3775879. Nilaratanakul, Voraphoj, Debra A. Hauer, and Diane E. Griffin...Griffin. "Development and characterization of sindbis virus with encoded fluorescent rna aptamer spinach2...et al. "Ribonucleoprotein purification and characterization using RNA Mango." RNA23.10 (2017): 1592-1599...

...start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid, while... highlighted below the table.     Figure 2: This decision tree can help guide your choice on ...transfer to the adult brain. Nature Biotechnology, 34(2), 204–209. https://doi.org/10.1038/nbt.3440 Goertsen...Thomson, M., & Gradinaru, V. (2021). Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional..., with significant contributions from Cynthia Arokiaraj. Addgene would also like to thank Viviana Gradinaru.../doi.org/10.3389/fimmu.2021.730825 Campos LJ, Arokiaraj CM, Chuapoco MR, Chen X, Goeden N, Gradinaru V...Adams, C. D., Yang, D., Wang, T., Wolfe, D. A., Arokiaraj, C. M., Ngo, V., Campos, L. J., Griffiths, J. ...

... 2008 Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, ...Tsiang H, Ceccaldi PE. Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: ...

...Reports 9: https://doi.org/10.1038/s41598-019-41088-2 Magnus C, Lee P, Bonaventura J, Zemla R, Gomez J, ...to one receptor, so it is important to fully characterize the compound, or compounds, you choose to ensure...

... 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces...

... the Cas machinery where to cut.      Fig. 2: Heatmap indicating loci where SNPs reside on sgRNA...from work being done today, is a nearly entirely separate effort from acknowledging its existence. Studying...

... replication competent virus [usually HIV-1]; and 2) the potential for oncogenesis through insertional... are technically gamma-retroviruses - another, separate member of the retroviral family. While both lentiviruses...

... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...adapted from Farahani et al. 2022, under CC-BY license.   Find pYtags plasmids here!   Farahani PE, et al...for monitoring specific RTK activation in vivo (Farahani et al. 2022). The authors take advantage of the...better response kinetics and photochemical characteristics than previous NIR calcium reporters iGECI and...   Matlashov ME, et al. Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium...for lactate developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key... here!   Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization...

..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is quantitative and...also collected data on fluorescent protein characteristics such as the brightness, photostability, maturation...Cranfill et al, 2016; Lambert, 2019). These characteristics are important, but have less priority than ...microscopy (University of Amsterdam). He develops, characterizes, and uses genetically encoded fluorescent probes...PMC4383620. Botman, Dennis, et al. "In vivo characterisation of fluorescent proteins in budding yeast."...brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry...

...Nature 339 (1989). 487–488. PubMed PMID: 2725683. 2. Miller, C.J., Kassem H.S., Pepper S.D., Hey Y., Ward... storage buffers used, and other experimental parameters. For example, while freeze thaw is generally ...25073060. 8. Finkelshtein, D., Werman, A., Novick, D., Barak, S., Rubinstein, M. “LDL receptor and its family...

... PMID: 9537413. PubMed Central PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating...and virtually never exceeds 40%. While this is comparable or better than the efficiency of most current...ribonucleoproteins (RNPs) that can be supplied separately from the viral vector. This delivery method frees...

...vectors with EYFP split at 175 Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components...

...rules are: (1) don’t use the same overhang twice; (2) avoid palindromes; (3) no overhangs with the same...highest-fidelity overhang set it can find for those parameters. For even more specificity, you can exclude specific...

...systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created with...been challenging. It can be difficult to ensure comparable expression of all components of Cascade for maximum...

...that view (Callahan et al., 2022).     Fig. 2: Students in an MDH CURE replaced select residues ...Part I – guided inquiry – purification and characterization of a fusion protein: Histidine tag, malate...

...interesting things to do, feel free to crowdsource! 2. Free food generally gets people to show up If you... company culture here at Addgene has remained paramount. Candidates who interview at Addgene frequently...