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We narrowed to 1,035 results for: URE

Showing: 301 - 320 of 1035 results

  1. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID...rough "COVID-19 effect" on order data, it does not capture the fact that different parts of the world encountered...='31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and...during weekends and significant holidays. Figure 4: Plasmid request trajectories suggest an exponential...data likelihood with multivariate Gauss-Hermite quadrature, since the analytical expression for the data...winter break, and COVID-19 on plasmid requests Figure 5: Inferred effects. Values are the inferred percentage...

  2. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...rate. A rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but...what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible? This...digital PCR involves partitioning a PCR reaction mixture into approximately 20,000 droplets using water-...containing thousands of droplets) can be sufficient to capture the information needed for your PCR experiment....droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor droplet...reader extracts the droplets from the plate and measures the fluorescence amplitude of each one. Droplets...

  3. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...plate to start liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you...use depends on your preferences. Either way, make sure to test your colony PCR primers before using them...will need to screen.   Saving clones for later culture: At this point, you will want to hang onto your...bacteria-water suspension and use them to start cultures of your positive clones. If you want to store ...ones. Regardless of which method you choose, make sure to use the appropriate antibiotic for selection.... on an agarose gel to determine their size. Make sure to use an appropriate molecular weight standard ...samples before pipetting them onto the gel. The figure above summarizes generalized expected results for...

  4. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ... Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche...is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat region...RNAs important for Cpf1 function, they found that mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides.... With this information, they turned to a cell culture system to see if any Cpf1 nucleases would exhibit... its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively...deletions at the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair...Mismatch positions are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength...

  5. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ... way to grow haywire, access and create more vasculature to feed themselves, use the blood stream to commute..., introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized...the host immune mechanisms altogether and escape future immune surveillance. Dormancy of certain cancers... system to the presence of cancer are unable to mature into effector cells in the tumor microenvironment... look at a variety of up and coming therapies. Future directions and open questions in cancer treatment...constantly in flux and is dependent on the selective pressures placed upon it by its environment and the body...immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949. PubMed PMID: 15322536...

  6. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide...transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More importantly...AAV or by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct...probe gene-function relationships in defined substructures of the CNS as well as individual neuronal populations...neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...

  7. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...identify a few of the features that confer broad host range in plasmids in nature, and provide tips on ... identified some of the features that confer broad host range. These features are: The plasmid origin...characteristics of natural plasmids and how they function in nature can inform us on how to create better recombinant...on how we can harness those features to create broad host range recombinant vectors.  A plasmid’s host...are versatile and adaptable. Variation in ori structure allows plasmids to utilize diverse host replication...and reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad host ...

  8. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...is to ensure the propagation of themselves and their associated genes. These systems better ensure that...that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems”...the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct... repeats. In cells where recombination has not occured, methanol induces the production of the toxic MazF...the mok SD resulting in a double stranded RNA structure that is recognized and cleaved by RNaseIII resulting...bioluminescence reporter in vivo in the absence of selective pressure and found that plasmids were maintained at 100%...with plasmid addiction systems While selfish in nature, these systems have been adapted by researchers...

  9. The Open Repository of CRISPR Screens: CRISPR Screen Data in One Place

    Type
    Blog Post
    ... identify. Figure 2: Users can cross-interrogate screens from the literature for a drug, disease...particular screens of interest.   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary...large-scale datasets by computational biologists. Figure 1: The BioGRID Open Repository of CRISPR Screens...compiling genome-wide datasets, which are often obscured in large supplementary data files, BioGRID ORCS.... Importantly, metadata for each screen is captured by BioGRID curators, including the cell line/tissue...determine the gene hits for each screen are also captured as metadata.   Screen results are reported as ...comprehensive pre-compiled downloads and via a full-featured REST Web Service API. Both options are fully open...

  10. Tips for Improving Your Next Manuscript

    Type
    Blog Post
    ... quality figure, produce the figure, its legend, and write a results section for that figure. Writing ...next manuscript 1. Be consistent with your figures The figures of a manuscript are the result of a lot of... to my PI about becoming a better writer, I was unsure if I should possibly take a composition class in... individual sections of a paper, the abstract, figures, introduction, etc. so as to thoroughly explain...quilt with your lab mates, you add the individual figures and results to make the final product with an interesting...Scientific Manuscript Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Write for the...

  11. Antibodies 101: Epitope Availability

    Type
    Blog Post
    ...assays, such as ELISAs and Westerns, frequently denature the proteins as part of the sample prep. This ...it from its native state to a less folded, or denatured, one. This enables absorbing of the proteins to... or ELISAs may therefore often be specific to denatured versions, or even to just that particular assay...    Fig. 1: A native protein (left) is denatured, allowing the antibody to bind to a previously...you’re working with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often... state or specific configuration. So how do you ensure that you have the right antibody for the protein...Did you have a denaturing step? Do you want to capture the protein bound to anything? Is it a transmembrane...

  12. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

    Type
    Blog Post
    ...marker and origin of replication. During bacterial culture, only the plasmid with the antibiotic resistance... that contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be...researchers find that using a 30°C incubation temperature to grow bacteria more slowly helps reduce the...Esposito (2018) noticed that in Stbl3 bacterial cultures with lentiviral vectors, the flat white colonies...tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone. Test multiple...multiple colonies before saving a glycerol stock to ensure the colony you selected contains the full plasmid...length plasmid. If you find that your bacterial cultures contain a mix of full length plasmid and the recombined...

  13. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... of these methods to ensure your insert retains the 3' A overhang: Use a mixture of proofreading enzyme...restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the...how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the blue PCR product...insert, the magic of TOPO cloning happens. Basic procedure Let’s break down the steps needed for TOPO cloning... TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction on ice if you are...extension time after the last cycle of PCR to make sure that the "A" gets added to all PCR products. Keep...electroporation to fail). When incubating at room temperature, it is not recommended that you exceed the 5 ...

  14. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ...mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038/nature.2016.19754 Zhang H, Demirer...Fan C, Landry MP (2019) DNA nanostructures coordinate gene silencing in mature plants. Proc Natl Acad Sci...sufficient to modify the genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting...components is essential for any CRISPR experiment, and failure to express either the gRNA or Cas9 in your cell...into a target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated ...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated... (carbon nanotubes, Kwak et al., 2019), DNA nanostructures (Zhang et al., 2019) or cell-penetrating peptides...

  15. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ... not being interviewed, there could always be a future boss in the room, so it is a good idea to start...academic position, see this very good article at NatureJobs: Interviews: The All-Important Job Talk. 1. Watch...Scientists do this all the time, you just need to figure out how to make it suspenseful. 4. Start preparing... Google.slides or Powerpoint file right now to capture interesting key results, striking images, creative...Of course, if some of your work does relate, make sure to include that material, but don’t force it. You...explaining how your work fits in the bigger science picture. Scientists especially like to learn new things...resulted from their research, “aha” moments, and future research directions. Don’t worry if your work hasn...

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

    Type
    Blog Post
    ...spectrophotometer measures these absorbances using UV-transparent cuvettes. First you measure the absorbance...in. This is a “blank” and measures the background absorbance. Then you measure the absorbance of the DNA...DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and whether...Generally, A260 of 1.0 is equivalent to 50 ug/ml pure dsDNA. Use the following formula to estimate your...as compared to spectrophotometric methods that measure all nucleic acids). These methods are more sensitive...quantified first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary...sensitivity so it’s not used much. However, the measurements are made in the visible range and can be read...

  17. Using CRISPR/Cas9 to Edit Disease Out of the Genome

    Type
    Blog Post
    ...absolutely possible to use CRISPR to cure genetic disease in the near future,” Li says, though there are some... derived from iPSCs. From Rohn et al. 2018; see figure 3 for full details.    CRISPR advances in monoallelic...receptor (CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic ...When all was said and done, the researchers had cured 24 mice of their disease. Meanwhile, Hans Clevers...because the genomes of adult stem cells in organoid cultures are stable,” said Gerald Schwank, first author...off-target effects is a major limitation. The procedure will also need to be perfected until the efficiency...of repair approaches 100 percent. “We need to be sure it's accurate enough and efficient enough for repair...

  18. Celebrate National Book Month with Addgene

    Type
    Blog Post
    ...Austen’s Pride and Prejudice, and some more obscure treasures, like Tove Jansson’s The True Deceiver. There...on-site book swap/library, reading is part of the culture here at Addgene. Since it’s National Book Month...own hands. Who doesn’t love a book series that features a young female protagonist, some clever magic,... may be intimidating, Ruthefurd’s writing will capture the attention of even the most skeptical reader...club Indeed, reading is ingrained into Addgene’s culture. We have a book club in the office that was founded... had a few reasons for launching this one. On a purely selfish level, I wanted to be challenged to read...has become one of my favorite parts of Addgene’s culture. Have you read any of these books? Whether you ...

  19. Addgene Depositors Get More Citations

    Type
    Blog Post
    ...our journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids... (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1...also pulled the big interdisciplinary journals: Nature, Science, PLOS One, and PNAS. We then broke the...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the... Addgene plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids...papers with and without Addgene plasmids are in Nature. This is also true for other journals, so our estimates...Environmental Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these...

  20. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...strand in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...presumptive mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction...Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered that the... of approximately 6 hours. To our surprise, we measured identical dissociation rates for catalytically...catalytically inactive dCas9 produced a “bubble” structure in uncut target DNA that was accessible to anneal...single stranded DNA. We thus wondered if such a structure could drive sequence replacement in cells. Targeting...inactive CRISPR-Cas9 using asymmetric donor DNA." Nature biotechnology (2016). PubMed PMID: 26789497.  2...
Showing: 301 - 320 of 1035 results