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We narrowed to 1,025 results for: URE

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  1. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...externally fertilized, transparent progeny (Figure 2). These features give it a leg up (or a fin up?) over other...become such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate... tracing in zebrafish To highlight some of the features that make zebrafish useful, I’ll outline two recent...of foreign DNA. Despite these caveats, several features of zebrafish—including the available genetic resources...compensation triggered by mutant mRNA degradation." Nature 568.7751 (2019): 193. PubMed PMID: 30944477. Farrell...cellular resolution in freely swimming zebrafish." Nature methods 14.11 (2017): 1107. PubMed PMID: 28892088...

  2. Plasmids 101: Shuttle Vectors

    Type
    Blog Post
    ... there. Make sure you read the instructions for whatever antibiotic you choose to ensure it works in your...shared by every living thing fit onto a single lecture slide. That shared language is the basis of shuttle...are designed to shuttle between species. Figure 1: Shuttle vectors are vectors that can function... your vector between, you should have a plan to ensure it persists in both contexts. For a vector ...This is useful for keeping your eukaryotic cell cultures bacteria-free, but it won’t allow you to select... producer cells, which have the machinery to manufacture infectious viral particles. Those particles are...

  3. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ... host cell's initiation proteins. In nature, such plasmids ensure their propagation by limiting their ...replication in bacteria is carefully regulated to ensure that it occurs at the appropriate time—for example...lower levels of DnaA to get the job done.   Figure 1: DNA melting by DnaA and IHF is the first step...Termination sequences present in the chromosomal DNA ensure that the helicases are released from the DNA once...of the chromosome, ending replication.   Figure 2: Prokaryotic chromosomal replication. Adapted...high, restraining further replication.   Figure 3: Comparison of replication regulation mechanisms...

  4. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    ...exploring nature in the real world. Additional Resources on the Addgene Blog Browse our CRISPR featured topic...frameshift or 21+ bp indel. This score is a useful measure to determine how many of the contributing indels...CRISPR-Cas9 double-strand DNA breaks using SpCas9. More features will be added to the ICE software tool soon. To... To request other nucleases or other features and functionalities, please email [email protected]. ...sequence for the nuclease used. Currently, ICE is configured for Cas9 fromStreptococcus pyogenes (SpCas9)....frameshift or 21+ bp indel. This score is a useful measure for those who are interested in understanding how...

  5. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...transducing your virus. Let’s start there!     Figure 1: Flowchart showing the process for using pooled...coli are allowed to grow on plates or in liquid culture to amplify the library, followed by DNA isolation... sequencing and further data analysis. Based on Figure 31.5.2 from https://pubmed.ncbi.nlm.nih.gov/27366891... and replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in...is controlled by viral titer, so you’ll need to measure your viral titer before you can transduce your ...with titer, cell type, and other factors, so make sure to put that aliquot to full use! Common conditions...that a cell doesn’t get infected at all. To make sure that every plasmid’s payload is adequately represented...

  6. Transferable Skills Guide: Career Planning Resources

    Type
    Blog Post
    ...or resume). If you receive a card in return, make sure to follow up with a professional “thank you” via...your CV doesn’t necessarily mean that you are a future employee of the company. It does, however, offer...to consider, some of the best options are the Naturejobs Career Expo and the ACS career fair, which are... candidates. If you do receive such a call, make sure to get that person’s contact information and again...when you are looking to change positions in the future. Anyone looking for work in today’s job market ...with you to ease the way. Make connections, and nurture those relationships. Then when you are ready to...job, you’ll be in the best possible position to secure it! Further Reading and Outside Resources http...

  7. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...for at least 12 hours, or at lower temperatures for longer. Cultures induced with arabinose and colonies...results in the production of air-filled protein nanostructures called gas vesicles, which scatter sound waves..., used in bacteria, expressed poorly at body temperature (37oC) and were too burdensome to express in ...Excess gvpA plasmid added into the transfection mixture in HEK293T cells gave drastic improvement in expression...transfection reagent (e.g. PEI-MAX). Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954...noninvasive imaging of microorganisms in mammalian hosts. Nature 553, 86–90 (2018). Farhadi, A., Ho, G. H., Sawyer...

  8. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038/nature14136 Pan J, Meyers RM, Michel...screens can be used to gain insight into protein structure and function (Pan et al., 2018). cDNA libraries... therapeutic targets using CRISPR–Cas9 screens. Nature 568:511–516 . https://doi.org/10.1038/s41586-019...C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional ...2018) Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness...Zetsche B, Volz SE, Zhang F (2015) A split-Cas9 architecture for inducible genome editing and transcription...

  9. Data Freedom: The Expansion of Data Sharing in Research Publications

    Type
    Blog Post
    ...datasets with their resultant figures. Instead of providing a “dead end” figure or table, clicking on the ... descriptions, legends and limiting numbers of figures such that many have multiple, complex panels. PLOS...sizes) but images should be unprocessed and in capture format. This raises another issue of proprietary...Other new data initiatives are also taking root. Nature Publishing Group recently launched a publication... and funders. Clearly, it is in our interest to ensure that our data are reproducible and useful to others... alpha isoform of GSK-3 to the same (deserved) stature as the beta isoform. Follow him on Twitter @jwoodgett...

  10. Addgene's Website Update

    Type
    Blog Post
    ... biggest change is to our most frequently used feature: the search bar. We’ve changed the colors and centered...to go straight to your materials of interest!   Figure 1: The new search bar.   Pro tip! You can use a...will prompt you to log in or create an account.  Figure 2: The new deposit materials bar New menu Most ...website without scrolling up and down the page.  Figure 3: The Education & Tools menu Further down What...look and a fresh new friend: Antibody of the Day!  Figure 4: The plasmid, viral prep, and antibody of the...t take our word for it — check out all the new features and designs addgene.org! And when you’re finished...

  11. Inntags: Innovative Protein Epitope Tagging

    Type
    Blog Post
    ... "Inntags: small self-structured epitopes for innocuous protein tagging." Nature methods (2015). PubMed...each tagged protein must be tested carefully to ensure it retains the attributes of the native protein...Georgieva et al. to develop new tags with defined 3D structures less likely to compromise protein folding and...Due to their limited effects on native protein structure, inntags may become the tags of choice for examining...correlation for protein localization in mammalian cells." Nature methods 10.4 (2013): 315-323. Pubmed PMID: 23435261...

  12. Let There Be LITE Plasmids

    Type
    Blog Post
    ...Addgene’s repository for use in neurons, both in culture and in living brain tissue. “The brain is such ... that’s exactly what the LITEs now enable. In a Nature paper introducing the optogenetic tool, Konermann...said in MIT’s press release. In an accompanying Nature News & Views Andreas Möglich and Peter Hegemann...of transcription occurs within minutes of light exposure. Third, the response can be graded with light ...applied non-invasively, its use is not restricted to cultured cells but extends to freely moving animals, as...endogenous transcription and epigenetic states." Nature. 500, 472–476 (22 August 2013).   , with the ...

  13. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...mammalian evolution. Nature 428, 493–521 (2004). https://doi.org/10.1038/nature02426 Iannaccone, P. M., ...processes and developments. Model organisms often feature genetic, physiological, or developmental characteristics...laboratory settings. One of its most attractive features is its stability and easily obtainable growing...axolotl has the ability to regenerate complex structures such as limbs, the spinal cord, and internal ...disease research. One of their most attractive features are their eggs. Frog eggs are large, opaque, and...genomics study in bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467...

  14. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ... 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...

  15. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...get several interviews but no job offers, is a failure to clearly articulate one's talents and ideas. ...Have a competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/...techniques. You DO need to be prepared to know the literature of your field and all of the caveats and alternatives...misfolded proteins is essential for human health. Failure to do so is implicated in several human diseases...much easier to focus on the questions when you’re secure in the knowledge that you have given everyone a...you want to convey for each section or aim. Make sure those get priority. The fine details for aficionados... is the task. Do your homework. Learn what your future potential colleagues study. Think about how you...

  16. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    ...four-stranded branched structures that form when elongation of the invasive strand “captures” and synthesizes...genome instability and contributes to cancer, premature aging, and other harmful processes. The most dangerous...strand; this results in the formation of a DNA structure referred to as the displacement loop (D loop)....and the resulting mutations after an HDR event, ensure the gRNA or PAM site is disrupted in the donor....engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013). PubMed. ...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about Non-Homologous End Joining...

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

    Type
    Blog Post
    ... 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...

  18. Management for Scientists: Seeking Feedback

    Type
    Blog Post
    ...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...

  19. Increasing Your Work's Visibility with Addgene: Citation, Search, and Collections

    Type
    Blog Post
    ... 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...

  20. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ...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...

  21. What's the Best Way to Elute and Store Your Plasmid DNA?

    Type
    Blog Post
    ...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...

  22. CASTing Off for New Shores in Human Genome Editing

    Type
    Blog Post
    ... 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...

  23. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

    Type
    Blog Post
    ...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...

  24. Oh, The Places You Can Go: Careers in Science Communication - Curriculum Development

    Type
    Blog Post
    .... 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...

  25. Oh, The Places You Can Go: Careers in Science Communication - Science Writing

    Type
    Blog Post
    ... 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 ...

  26. Bricking Science: Portraying Scientific Reality Through LEGO

    Type
    Blog Post
    ...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...

  27. Management for Scientists: Managing vs. Leading

    Type
    Blog Post
    ...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...

  28. 5 Uncommon Ways to Gain Career Skills During an Uncommon Time

    Type
    Blog Post
    ... 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 ...

  29. Viral Vectors 101: Parts of the AAV Transfer Plasmid

    Type
    Blog Post
    ...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 ...

  30. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    ...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...

  31. What Do I Do Now? Academic v. Non-Academic Career Decisions

    Type
    Blog Post
    ...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...

  32. Viral Vectors 101: Transductions

    Type
    Blog Post
    ...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...

  33. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    ...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...

  34. Plasmid Cloning by PCR

    Type
    Blog Post
    ...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...

  35. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    ...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 ...

  36. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    ...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...

  37. Improving Transduction Efficiency with Exosome AAVs

    Type
    Blog Post
    ...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...

  38. Hot Plasmids - January 2023

    Type
    Blog Post
    ...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 ...

  39. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...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...

  40. What Good Citizenship Can Do for Reproducibility in Science

    Type
    Blog Post
    ... 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...

  41. Components of CRISPR/Cas9

    Type
    Blog Post
    ...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,...

  42. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    ...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...

  43. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ...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...

  44. Hot Plasmids - May 2022

    Type
    Blog Post
    ...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...

  45. Antibodies 101: Monoclonal Antibodies

    Type
    Blog Post
    ...focus on antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called HAT. This medium selects...antibodies are particularly useful when you want to ensure that the antibody binds to a specific region of... during your experiment, then you’d want to make sure that you choose an antibody that recognizes an epitope...polyclonal antibodies - which are a heterogenous mixture of antibodies that recognize many different epitopes...plasmid to create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes...

  46. Q&A with Karen Katz and Carl Paratore, the Newest Members of the Addgene Board of Directors

    Type
    Blog Post
    ...important considerations for the future of open science? What about the future of open science are you most...do you hope Addgene will accomplish in the near future? Katz: I’m excited that Addgene is continuing to...those “what could go wrong scenarios” and making sure we have adequate process and controls in place to... particular area or profession, you need to make sure you have learned and mastered the fundamentals.   ...was a founding member of Mass Medical Angels, a venture mentor at MIT and served on the Boards of BioBuilder...

  47. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    ...cell line cultures. Lentivirus Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus... gamma-retrovirus including genome architecture, host integration, and its association with disease. The...cell types. Altogether, AAV has many excellent features for human gene therapy and is currently being ...types of interest. This is a particularly nice feature as organ-specific injections, or the location of...   Table 1: Types of viruses and their features Choosing a virus Now that we’ve reviewed the...

  48. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ...provided by the manufacturers and enables customized experiments. Projects such as the OpenFlexure Microscope...modify the device for individual experiments. Manufacturers rarely provide the construction plans or CAD...accessible open-source hardware. Projects such as the OpenFlexure microscope or open-source modular optical toolbox...documentation, which facilitates the reproduction of the structure in a similar way to the biological protocol, and...et. al, "Robotic microscopy for everyone: the OpenFlexure microscope," Biomed. Opt. Express 11, 2447-2460...

  49. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...negative (digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target ...precision. This means digital PCR provides an absolute measure of target abundance, does not require standard ...directly at the predicted double strand break site (Figure 2A). The single molecule resolution of digital...completely “blind” to this very important difference (Figure 3). If you are interested in harnessing the power...requires target DNA sequencing to confirm the exact nature of your mutations. A great description of the plethora...induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–826 (2013). PubMed PMID: ...

  50. A Novice's Perspective on Neuroscience - SfN2017

    Type
    Blog Post
    ...scientists and nonscientists alike). My only in-depth exposure to neuroscience prior to this conference was a.... This was not at all the case. The conference featured people who work in sociology, behavior, and artificial... session organized by Story Collider and which featured a talk about the different parts of the brain ...getting into neuroscience from the mol bio side you’re sure to have many opportunities to learn about and develop...website and on the Addgene blog to get some ideas for future experiments and to find tools that will make your...Ed Boyden on Optogenetics, Neuroscience, and the Future of Neuroengineering Primer on Optogenetics Resources...
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