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We narrowed to 1,048 results for: nin

Showing: 301 - 320 of 1048 results

  1. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ... nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally... Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected and buffer exchanged...titering by qPCR quantifies the number of genome-containing viral particles that are present in an AAV prep...standard curve, which is generated from an ITR-containing plasmid. This whole process takes ~3 hours to...expression in mammals Use the PEI calculator for planning AAV packaging transfections Find tips for a first...

  2. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    ...you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half...microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the...solves most problems before they occur. With proper planning, you’ll know exactly what materials you’ll need...changes in temperature (especially if you have air conditioning on) so leave them in a sterile incubator. This...make up your master stock, while you can use the remaining vial to grow and freeze additional cells that ... Universität Berlin, Germany. She will soon be joining her PhD lab at the Max Planck Institute of Colloids...the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

  3. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...were used in this study.  The zCas9i cloning kit is a plant cloning kit that contains a series of vectors...Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based vaccines...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...protein VP1 is co-expressed with a fusion protein containing VP2C and a protein of interest (POI). The VP2C...with more on the way. S5E2 regulatory element containing vectors developed in the Jordane Dimidschsteind...provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...

  4. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    ...Neurological Surgery at the University of Miami. The beginning of this century has seen some major advances in...originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis...confocal microscopy In confocal microscopes, optical sectioning of a specimen is based on discriminating the ...to photobleaching.  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow...photo-bleaching or photo-damage than in conventional laser scanning microscopy . Since image acquisition is performed... of imaging rodent brains and spinal cords by combining Ultramicroscopy with tetrahydrofuran-based tissue...Leischner, A. Schierloh, N. Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed...

  5. Sharing Your Lab Protocols: Using Apps to Save Time & Track Your Experiments

    Type
    Blog Post
    ...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...scientists, publishers, and small start-ups are turning to software apps to solve our need for quicker,...protocol listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene...has also cataloged a variety of useful molecular cloning protocols, including making LB agar plates, restriction...labs, and now Addgene. I just began uploading our cloning protocols to the site and will keep you updated...

  6. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    ...loss of function meaning that if an essential gene plays a role in the desired screening phenotype, it may...knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives....copy number dsDNA cutting. A Dolcetto library containing only 3 sgRNAs (setA) also outperformed CRISPRi...conducted with only two constructs (the other containing dCas9-VP16). Like CRISPRi, CRISPRa is only effective...the SAM library approach in a comparison study screening for vemurafenib resistance in A375 cells. The ...screens Read this blog post about genome-wide screening using CRISPR Resources on Addgene.org Find CRISPR...

  7. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ...so that you can specifically isolate bacteria containing your constructs of interest. But does it matter...will lose it during division. These non-plasmid-containing bacteria can compete with and confound the goal...from forming while effectively selecting plasmid-containing bacteria. So… why do some people still use amp...? Zeocin is genotoxic when not 100% inhibited, meaning it will induce mutations in the host/plasmid DNA...Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set="fontawesome-5.14.0" name...References and Resources References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks...Antibiotic Resistance Plasmids 101: Blue-White Screening Choosing Your Perfect Plasmid Backbone  ...

  8. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...research interests include proteomics, machine learning, and visualization for big biology. GPS for the...1, 10) indicates a stretch of DNA at the very beginning of the third chromosome, ten base pairs in length... us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging family of "...all of those 13,000 regions are important for maintaining ESCs. So which of these 13,000 regions are critical...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...Lentivirus from Addgene If you’re interested in designing your own CRISPR screens for enhancers, check out...database. Aneesh's research interests span machine learning, proteomics, and user interfaces for math.     ...

  9. Negotiating Work and Life: How to Find the Joy

    Type
    Blog Post
    ... on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...If you enjoy working and come home happy in the evening, your friends and family will truly sense and appreciate...within these bounds You can work later (in the evening, or in a few years) when life responsibilities ...Someone else can clean your house, pick up your dry cleaning, cook your dinner and deliver your groceries. ...nights when you can’t get home, babysitting or running errands. Give help when you have time, and in return... things can’t. I kiss my kids good night every evening I am home no matter how busy I am. Believe it or... really busy, but I go to the gym almost every morning. Of course, this is more than just fun, as your...

  10. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    ...after negative staining. Empty vector particles can be identified after negative staining and appear darker...Physical titer: the concentration of viral particles containing viral genomes. Physical titers are measured by...full to empty viral capsids: the ratio of genome-containing viral particles relative to the total number ...approximately 50% of viral capsids were genome-containing (“full”) (Zeltner et al., 2010), while another...Grimm et al., 1999). The percentage of genome-containing viral capsids is typically quantified by electron...representative viral vector preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph...

  11. Tips for Writing a Good Cover Letter

    Type
    Blog Post
    ...letter, they have an opening, a body, and a closing. Parts of a cover letter The opening paragraph is the... by giving specific examples of your work and explaining how your experience and skills would help you...application. These could be logistical, like briefly explaining a gap in your job history or sharing relocation...your organizational system, your rationale for designing it the way you did, and state that it would help...how you feel connected to their work. Since the beginning is often the hardest part, here are some examples... in [specific topic] and in the possibility of joining your team as a postdoctoral researcher. Dear Hiring...

  12. How Exercise Purges Stress and Keeps Depression at Bay

    Type
    Blog Post
    ...muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out... acid. That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering... lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

  13. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    ...plasmids String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan...Read the paper in Genome Biology Find plasmids containing these base editors Spatially and temporally...gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one overnight reaction... Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi experiments...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...

  14. Quick, Versatile Plant Transgenesis with GreenGate Plasmids

    Type
    Blog Post
    ...introduced what’s been called a major breakthrough in cloning technology with the Golden Gate method. For the...researchers working in plant model systems. “The cloning is all done in vitro; it’s no different in mouse...to more tedious and time-consuming traditional cloning methods, Lohmann said. The key component of Golden... says their lab learned the hard way after the cloning method, which had been humming along at 99 percent... "GreenGate - A Novel, Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12)... Engler et al. "A One Pot, One Step, Precision Cloning Method with High Throughput Capability." PLoS ONE...

  15. March for Science

    Type
    Blog Post
    ...however, inconvenient data must not be ignored. Returning to an earlier analogy, no amount of shouting will...scientists have chosen to leave the lab and march. Unintended consequences While frustration and anxiety have... science, others are concerned about possible unintended consequences of a march. Three of these risks...of everyone in government (19). To defend the meaning of the word ‘fact’. Policy must be based on our...science advocacy opportunities. Information for joining the march: March for Science: The website for...For science to be integrated into politics in meaningful ways, continued conversation about science’s ...: Universities Report Fourth Straight Year of Declining Federal R&D Funding in FY 2015 National Science...

  16. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...dynamics in live mice under a variety of behaviors, opening up new research directions that were inaccessible...concentrations in cells than existing sensors. Using genome mining and structure-guided engineering of potassium binding.... Figure 5: Structure-guided design and genome mining both led to new sensors with lower binding affinities...sensor plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent... Indicators through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022 May 27;7(5):1336-1346...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

  17. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein...please write to us in the comments section.  FX cloning kit This kit is designed to be used with Type IIS...sites used in these vectors are of low abundance (meaning they are unlikely to be found in your gene of interest... incorporation of extensive sequences used for cloning into the final translated products. Negative selection... help decrease false positives observed in the cloning process, making it easier to produce the constructs...interest in a variety of model systems, thereby opening your research to a variety of experimental opportunities...fusions all use the same restriction enzymes for cloning. Furthermore, some plasmids contain peptides to...

  18. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...dividing and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral vectors...between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...transfected into cells as a donor plasmid containing the transposon, along with a helper plasmid that expresses...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...help mediate their transduction, such as the polyarginine peptide described in Zhou et al and Kim et ...large nucleoli, and little cytoplasm. Positive staining for alkaline phosphatase, expression of embryonic...

  19. Deep Dive: qPCR

    Type
    Blog Post
    ...appropriate choice (Panina, 2018). Look for studies specifically aimed at determining the best options for...Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-... amounts of reagents in your chemistry, unless running a multiplex reaction or on a rapid cycle machine... quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...Digital Droplet PCR for AAV QuantificationPlasmid Cloning by PCR...

  20. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...contain BsaI sites for easy cloning of a gRNA of interest. Cytidine and adenine base editing in Streptomyces...breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many...applications for which they’ve been used. The beginnings of bacterial CRISPR engineering One common way...template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria are more recombinogenic ...directly modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg ...inducer, in this case anhydro-tetracycline (aTc). By cloning in two tandem copies of the gRNA cassette, one ...Sun lab demonstrates the utility of cytidine and adenine base editors that enable C-to-T and A-to-G base...
Showing: 301 - 320 of 1048 results