We narrowed to 1,042 results for: URE

... each requires selection. These two steps are (Figure 1): The initial plasmid integration step (the ...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which...for the loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is... of two visible blue or magenta chromoproteins (Figure 2) (Liljeruhm et al., 2018). This system is a nice...aeruginosa genome with two-step allelic exchange." Nature protocols 10.11 (2015): 1820. PubMed PMID: 26492139...of bacterial genomes using CRISPR-Cas systems." Nature biotechnology31.3 (2013): 233. PubMed PMID: 23360965...

... just hides in latently infected cells. To truly cure HIV-1, researchers need to vanquish these hidden...for HIV-1 patients. However, ART is not an HIV-1 cure. In patients who stop ART, viral levels will soon... the percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell...target sequence. Kaminski et al. next tested their procedure in HIV-1 infected T-cells to see if they could...therapies target HIV-1 at the level of protein structure, CRISPR gRNAs require DNA sequence-specific binding... note that these experiments were done in cell culture, and the cells infected with HIV-1 only after stable...papers present tantalizing evidence that an HIV-1 cure may be within our reach. Similar studies have shown...

...be boiled down to one, key concept: Creating a culture of excellent, effective communication between all...on the team talks and listens in roughly equal measure, keeping contribution short and sweet. One key... face one another, and their conversations and gestures are energetic.  This can take practice and training...training. This is important because it creates a culture of engagement in the issues at hand and eagerness...tactics for effective delegation will be covered in a future post in this series). Members carry on back-channel...talking about gossip here – this means encouraging a culture of maximum information flow so everyone knows what...

... you and the original depositor. Our citations feature makes it so that, when you cite a plasmid from ...to find your work later. We also hope that this feature will decrease redundancy. If you modify a plasmid...section. We’ve also added the Plasmid of the Day feature to our home page - each day we showcase a new plasmid...plasmid that you might otherwise miss. We hope this feature will draw attention to the great variety of plasmids...repository. And last, but certainly not least, we often feature new plasmid technologies on our blog and encourage...Evolving Check out the Blog Post on Our Citations Feature Browse More Inside Addgene Posts Additional Resources...

... the course of evolution, Tn7-like transposons captured and repurposed nuclease-deficient CRISPR-Cas systems...deliver large sequences and even entire genes. Figure 1: Mechanism of CAST systems. A Cas effector (Cascade...Natural transposons evolve under contrary selection pressures: high efficiency helps them spread, but also stresses...efficient CASTs (Witte et al., 2025). The best evoCAST featured 20 mutations in TnsB, the subunit that catalyzes...engineering using CRISPR-associated transposases. Nature Protocols, 19(3), 752–790. https://doi.org/10.1038...-Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://doi.org/10.1038/s41586...

...of time, especially at room temperature or at 4°C. Storing DNA: temperature and longevity You should consider...the DNA. pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors...are pH and DNase contamination.  Extreme pH can denature, break, and even change the DNA sequence. High...elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you choose...Plasmid DNA can be stable at 4°C or even room temperature for a short period, and there are indications...

...not terribly problematic when working with cell culture, because it is easy to harvest large quantities...sheering of DNA by sonification. Though it helps capture weaker or more transient DNA-protein interactions...of interest. First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads...thus targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...desired. It also provides an excellent additional measure of DNA binding sites in combination with existing...fusion proteins Study chromatin interactions with CAPTURE Additional Resources on Addgene.org Find simple...

...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera: ...taking many pictures, one for each position in a movement, and assembling the many pictures together to... our free time. After taking almost a thousand pictures, my husband Enzo will spend a few days editing...you an idea of the amount of work, see how many pictures we take to make a walk cycle of one step. Challenges...

.... And make sure to subscribe to the Career Channel of the blog so you don’t miss the future posts in this...eScience Labs.  “We work with faculty members to make sure the courses encompass what they want to teach.” ...onsite, Caitlin can visit the operations team to make sure “that everything that’s going into the kit is going...manager also requires Caitlin to look at the big picture: “It’s been really cool to take a step back and...science. And I do it well.’” Stay tuned for the next feature in this series. We’ll be interviewing Susan Keown...

... comments. They could be featured in future career blog posts. And make sure to subscribe to the Career...Iowa, he was like a lot of newly minted PhD’s: not sure what his next step should be. He debated taking ...few other tips: “Learn HTML, write a lot to make sure you don’t hate it, and be flexible. The best stuff...appealing images and colors draw people in. Drawing a picture of a mechanism that also looks nice is a great ...

...hire and fire to make sure the team is the best it can be. Managers must ensure that their teams are working...presentation many times, I could never actually figure out what skills were really leader-specific. How...doer mode even late in our careers.  Managers make sure the work gets done Managers do this by removing ...about her management style. Leaders look to the future Leaders must get new things started and then step...the driving inspiration, to create a productive culture and to correct systemic problems. Understanding...

...viral-mediated. Transformations are usually bacterial in nature. All three of these methods involve DNA incorporation...be flanked by viral packaging elements that will ensure the virus gets produced, and viral particles are...functional viruses later on. These built-in safety features provide extra layers of risk management when working...floating in it, and then sterile filtered to be extra sure nothing untoward gets transduced (like a rogue HEK...virus, cell type, and/or desired event.   Figure 1: Transduction to expression process. A viral ... the right method for your experiment.  A cool feature of viral work is that you can swap out your virus...

... foreseeable future and is a great way to get ready for the live audiences in your future.     3. Start...months, you may be looking for more ways to make sure you are moving forward. Here are a few out-of-the-box...yourself. You can watch yourself to critique and figure out what to improve. You can share it with others... The conversation builds a relationship for the future and the 1:1 format is good for everyone including... both building relationships and thinking about future career options. 5. Write a grant - even if you ...

...inherently unstable due to their secondary structure, palindromic nature, and high GC content. This instability...plasmid and take a look at each of its parts. Figure 1: Parts of an AAV transfer plasmid. Parts shown... sequence through ITRs due to their secondary structure and high GC content, but some companies now offer...spatial and temporal activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck ...Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which aids in nuclear export ...

...or by sequencing the unique DNA barcodes. Figure 1: Schematic of barcoding with a pooled library...genes that underlie a particular phenotype. Figure 2: Schematic of CRISPR knockout screening with ...accounts for chromatin, position, and sequence features in the selection of sgRNAs. Other CRISPR libraries...about viral evolution and antibody binding. Figure 3: Schematic of phage display screening with a ...libraries? Addgene’s pooled library collection page features hundreds more libraries to browse. Our Guide to...Ordering” to see what options are offered. Figure 4: Screenshot of the Addgene website. Let us...

...the extremely long, highly structured HCV genome (~9.6 kb of highly structured template!). MarathonRT also...localization in future studies of other genomic loci. Find the C-BERST plasmids here. Gao at al. Nature Methods...involved in transcriptional regulation and chromatin structure at your locus of choice. Find the Tet-inducible...Tet-inducible caspex plasmid here. Myers SA et al. Nature Methods. 2018. PMID: 29735997 bioRxiv preprint posted...

...career outside the traditional academic silo to tenured professor.  Non-academia encompasses millions of...he would give up,“an excellent—some say “cushy”—tenured faculty position for the grind of corporate life...industry but the “product” is publications—the pressure to produce is the same You get to interact with...business hours (mostly), but can often achieve more structured time management conducive to work/life scheduling...short and exciting ride Complicated matrix structures are common Small integrated teams, little hierarchy...interpersonal issues (one bad apple can change entire culture) Specific requirements for hiring more common...

...cleavage in mammalian cells, likely due to high temperature requirements. To solve this problem, Gao et al...NgAgo head to head NgAgo has a number of unique features compared to Cas9. These include: More targeting...vitro transcribed, and changes in gRNA secondary structure can affect editing efficiency. Gao et al. tested...1228 or 1307 aa depending on the species). This feature is advantageous if you’re working with small capacity...excision would alter the target sequence and preclude future HDR, which could lower HDR efficiency. Higher sensitivity...DNA-guided DNA interference by a prokaryotic Argonaute.” Nature. 507(7491) (2014):258-61. PubMed PMID: 24531762...

...but you should ensure that the bases do not result in the formation of a hairpin structure within your primer...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer...The process is shown graphically in the following figure, in which we add EcoRI and NotI sites to Your Gene...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length...

...cellular events such as the dynamics of subcellular structures and gene expression. You must see the movies ... a compatible fluorescent protein. FRET can be measured by exciting the most blue-shifted fluorescent ...optogenetic receptor, melanopsin, requiring brief exposure to 470nm blue light. To avoid overlap with this...gene tagging using a ligase-4-dependent mechanism. Nature Comm. 7, 12338. PubMed PMID: 27465542. PubMed Central...microscopy and enhances bioluminescence imaging in vivo.Nature Biotechnol. 34, 760-7. PubMed PMID: 27240196....luminescent protein for real-time multicolour bioimaging. Nature Comm. 7, 13718. PubMed PMID: 27966527. PubMed Central...NanoLuc-based luminescence (BRET) Ca++ sensing. Nature Comm. 7, 13268. PubMed PMID: 27786307. PubMed Central...

...and cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR Csm plasmids...lagged. Fixation, chemical staining, and light exposure can negatively affect FPs during these processes... also withstands denaturing conditions, high temperatures, and other chaotropic conditions better than...these preparations. After crystalizing the hfYFP structure, the group rationally designed GFP variants with...fluorescence in high molarity GdnHCl, at high temperatures, and in the presence of oxidizing agents. Adapted...Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids here!   Campbell, B.C., et ...

...the QCRG to continue our work toward a cure for COVID-19, be sure to follow us on Twitter (QBI_UCSF and...do anything and everything we can to help find a cure -- and quickly. Normally drug discovery is a process...much faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day...want to be careful with these clones (and help us figure out what is going on there!).  Find the Krogan ... be working on this problem more quickly. Figure 2: Getting our research in line, with a row of ...

...ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents...correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol ...al, 2012; Lawler et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high...Athersclerosis 2012. Pubmed PMID: 23068583 Thierer et al, Nature Communications 2019. Pubmed PMID: 31366908 Adapting...CRISPR activation plasmids at Addgene. Liu et al., Nature Communications, 2019. PubMed PMID: 31451697. Genetically... and offers many advantages over traditional measurements such as patch clamp recordings. However, technical...

... of being a “good scientific citizen” and making sure you do what you can to help your fellow researchers...subtle, and it can take a lot of detective work to figure them out. But, when they come to light, it’s clear...way so other researchers can use them, to making sure that methods and protocols are available and detailed...Press we developed STAR Methods to improve the structure and transparency of methods sections, including... room to do more. If we could really instill a culture of rewarding good scientific citizenship in all...making it happen would increase dramatically. I’m not sure what the best approach to doing that will be, and...

...and the guides are relatively unstable.   Figure 1: CRISPRainbow labeling. Two MS2, PP7, or boxB...interest, the team could visualize three loci and measure inter-locus distances. They tested this system ...intergenic DNA region (IDR3) as a reference loci to measure inter-locus distances of loci pairs from intergenic...variation (CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) Diagram...dCas9 and engineered sgRNAs using CRISPRainbow.” Nature Biotechnology 34 (2016): 528-530. PubMed PMID: ...scaffolds for signal amplification in genome imaging.” Nature Methods 15 (2018): 928-931. PubMed PMID: 30377374...

...are secreted from the cellular membrane into the culture medium. AAV particles are either associated with...vexosomes (vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles...differential centrifugation steps to separate cell culture supernatant containing exo-AVVs from producer cells...then to enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation...the production of exosomes or stabilize their structure or their interaction with AAVs (Schiller et al...model of glioblastoma (Volak et al. 2018). The structure of exosome AAVs is related to the use of microvesicles...

...cleave them leaving mature crRNAs bound to the Cas9/tracrRNA complex. D) The mature crRNA is used to guide...GAA, or GAT SpCas9-NG 3' NG  Staphylococcus aureus (SA); SaCas9 3' NNGRRT or NNGRR(N)  Acidaminococcus...the crRNA to Cas9 and facilitates processing of mature crRNAs from pre-crRNAs derived from CRISPR arrays...unique stretches of DNA between them “spacers” (see figure below). After years of research, we now know that...sequences required for processing pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions,...

...phosphorylation, promoting transcription (see figure below). Subsequent exposure to red light rapidly deactivates ...limited studies and can fluctuate with changing culture conditions. The use of low intensity light solves...promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates the system. Version...systems The large dynamic range of these systems ensures that they can be used with many types of proteins...Scouras A, Ellington AD, Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the...

...understanding the brain’s function is knowing its architecture, in particular the connections between different... uptake of these dyes in vivo, microinjection, pressure injection or iontophoresis (applying small electric...of RABV. This improvement resulted in a clearer picture of neuronal connectivity in the mammalian brain...but overall with rather low efficiency. Not all measures of transport efficiency are consistent which may...observations like these may be a starting point for future developments in the field of AAV vectors. Conclusion...achieve transgene delivery by targeting peripheral structures (such as injection into muscle) as opposed to...(2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed PMID: 19578477. PubMed...

...powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview). Until...doi.org/10.7554/eLife.64631  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... applies both positive and negative selective pressures for novel capsids. In a continuation of that work...Find M-CREATE plasmids here!  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... factors, including limitations of number of flourescent proteins that can be inserted into a cell without...visual barcode plasmids here! Kaufman, et al. 2022. Nature Comm.https://doi.org/10.1038/s41467-022-30008-0...