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We narrowed to 587 results for: des.1

Showing: 261 - 280 of 587 results

  1. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    Published
    Oct. 27, 2015, 3 p.m.
    ...sarcoma-associated herpesvirus.       References 1. Snapp, Erik. "Design and use of fluorescent fusion proteins ...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers ... disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation... live cells in real time. Previously, we have described a number of practical considerations that should...

  2. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    Published
    Sept. 7, 2017, 12:17 p.m.
    ... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...investigators to identify specific clones with desirable properties, either initially, or using secondary...

  3. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    Published
    Aug. 23, 2016, 2:30 p.m.
    ...everything I allow 1 slide for every 1 minute of talk length, and then I cut a few more slides. Do not run over...NatureJobs: Interviews: The All-Important Job Talk. 1. Watch other people give great talks We are not talking...suspense. Notice the simplicity of their slides and how the slides complement the spoken message. Learn from...speak to start creating slides or thinking about content. Create a Google.slides or Powerpoint file right...how these speakers talk without looking at their slides or notes. Listen to their stories and narratives...listeners to be thinking “What happens next?”  Describe how you pushed to a solution or overcame obstacles...term, but are worried that this might sound condescending, consider explaining the term on a slide but...

  4. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...guide RNA (sgRNA), provided it meets two conditions:  1) The sequence is unique compared to the rest of the...bystander bases” if you choose this system.  Fig. 1: Cytidine deamination takes place on the free strand...temperature-controlled genome editing. Nat. Commun. 2017 81 8, 1–9 (2017).Walton, R. T., Christie, K. A., Whittaker...vectors and design your sgRNA targeting sequence using one of the many freely available sgRNA design programs...many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single...Cas9 localizes the deaminase to where the edit is desired, and it performs the programmed edit within the...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...

  5. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from...In vitro screening for the best gRNA Figure 1: Validation of double strand break (DSB) mediated ...and greatest in scientific content.      References 1. Cho, Seung Woo, et al. "Analysis of off-target effects... screen them for your desired edit. Good news! Remember the primers you designed to generate your pCAG-EGXXFP...CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase...that do it for you. One such option is the CRISPR Design tool supplied by the Zhang lab. This simple website... and cons of using many different types of gRNA design software. When you are happy with your selection...

  6. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    Published
    Jan. 26, 2023, 2:15 p.m.
    ... to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs)....Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.  Additional Resources on the Addgene...your desired edit are all you need to generate a knock-in.   General considerations for designing a repair..., and ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one...increase the frequency of edits, researchers have designed self-cleaving plasmids that liberate the targeting...information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles driving CRISPRs...

  7. Twenty Years of Sharing: Addgene's Viral Vector Service

    Type
    Blog Post
    Published
    July 30, 2024, 1:15 p.m.
    ...catalog had grown to 879 viral catalog items (Figure 1). The service caught on quickly in the scientific ...over 100,000 viral vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene ...Neuromethods, 195, 77–101. https://doi.org/10.1007/978-1-0716-2918-5_5 Haery, L., Deverman, B. E., Matho, K...site, and titer. Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult...which include calcium and voltage sensors, are the broadest category of viral vector tools, which may help... for vectors in the “controls” category, which includes things like viral vectors that deliver GFP. ...most requested serotypes (Figure 4), our catalog includes vectors in a variety of serotypes, including the...

  8. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    Published
    Sept. 1, 2015, 2:30 p.m.
    ...your budget, you should take the following steps: 1. Define your timeline Start with your thesis in mind...these decisions in the following order of importance: 1. First make decisions based on experimental timelines... several factors aren’t taken into consideration. 1. Always focus on the science When negotiating finances...Wilpitz founder and lead consutant at Experiment Designs Consulting, Inc. “How much do you need [for your...necessary resources. This is why at Experimental Designs Consulting (EDC), we instead have our clients focus...the institution/funding source. Major equipment includes large centrifuges, sequencers and analyzers, and... as many of these tools as possible, this even includes clocks, timers, and phones.  As for reagents and...

  9. Year of the Camelid: Antibody Style

    Type
    Blog Post
    Published
    May 9, 2024, 1:15 p.m.
    ... similar antibodies in llamas and alpacas (Figure 1). At the time, researchers were already on a quest...nanobodies (Arbabi-Ghahroudi, 2017)!      Figure 1: Comparison of a heavy-chain only antibody and single-domain...camelids too, though not just for the reasons the UN describes. In honor of the Camelid family, I wanted to take...of times on this blog over the years and have described how they compare to other antibodies and affinity...

  10. Hot Plasmids - October 2022

    Type
    Blog Post
    Published
    Oct. 6, 2022, 1:15 p.m.
    ...protein sequence of the predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized ...several other constructs in plasmid form. Fig. 1: Summary of the approach and outcomes of the screen...optimized for 2-photon imaging of voltages. This includes monitoring of fast bursts, as well as slow up/down voltage...KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors show that...

  11. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    Published
    Jan. 30, 2014, 3:29 p.m.
    ...into E. coli is a fairly inefficient process– just 1 out of 10,000 cells on average! Without some means...down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite ...when the cells are grown on selective media, and provides those bacteria with a pressure to keep your plasmid...gene and acts as a marker for the mutation. When designing these types of experiments it is best practice...

  12. Tips for Getting a Faculty Position

    Type
    Blog Post
    Published
    July 20, 2023, 1:15 p.m.
    ...Full "Applying for a Faculty Position" eBook Here 1. Have a competitive CV You don't necessarily need ...that people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly... What will be the focus of your first grant? (1-2 sentences) Simply stated, what are your aims...Applying for a Faculty Position: the View from Both Sides," are summarized below. Check Out Erik Snapp's ...You want people to focus on you and your ideas. Slides will be     distracting. What is the big question...feedback. Many of these documents are too vaguely described in other sources of guidance or the document examples...single malt whiskies, but I remember the days of modest postdoc salaries all too well. If there's a group...

  13. History of CRISPR Cas - A tale of survival and evolution

    Type
    Blog Post
    Published
    Nov. 25, 2013, 10:12 p.m.
    ...sequences were initially discovered in E. coli in 1987 (1), the concept that these clustered repeat sequences...neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the...multiplexibility in various species and describe an assortment of applications. Despite the ubiquity of CRISPR/Cas ... store a record of invading phages but to also destroy the phage upon re-exposure (2, 3). More specifically...complex binds to the foreign DNA, a cut is made to destroy the invader. Wikipedia, accessed 25 November...engineering. The diversity of the CRISPR/Cas systems provides powerful lines of defense against invading phages...CRISPR/Cas inspired genome editing was initially described by Cong et al. and Mali et al. in January 2013...

  14. Plasmid Cloning by PCR

    Type
    Blog Post
    Published
    March 29, 2016, 2:30 p.m.
    ...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...for Gibson assembly. The steps following primer design and the PCR process itself are very similar to ...the downstream steps. For this example, we will describe how to copy a cDNA from one vector into a new ...YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning     The basic...: Do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...

  15. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    Published
    May 23, 2023, 1:15 p.m.
    ...et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches... Brewer, M., McDonough, L., Zhu, Y., Neumann, E., De Caestecker, M., Gutierrez, D., & Spraggins, J. (2022...). Springer New York. https://doi.org/10.1007/978-1-4939-8935-5_26 Jambor, H., Antonietti, A., Alicea,...between the targets. Or, it might be sufficient to describe or classify the labeled structures visually; for...will vary depending on your specific experimental design. Like selecting an antibody, some of the requirements...

  16. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    Published
    Sept. 1, 2020, 1:15 p.m.
    ...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer...doi.org/10.1002/emmm.201302948 Trapani I, Toriello E, de Simone S, Colella P, Iodice C, Polishchuk EV, Sommella...physical size of the genomes is still ~4.7kb. But despite this, larger functional transgene products were...HDR event can’t occur. Lastly, depending on its design, donor A may be capable of expressing a truncated...

  17. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter...lentiviral transduction. Lysine-specific Demethylase 1 (LSD1) Tatjana Sauka-Spengler's lab has deposited ...oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene 34...which uses light to regulate transcription, also includes light-regulated histone methyltransferases and...histone modifications. Like the TALE-LSD1 system described above, dCas9-LSD1 inactivates targeted enhancers...certainly not the only CRISPR-based technology designed to alter gene expression. Fusing dCas9 to a transcriptional...epigenetic modifiers? As with any experiment, your desired outcome will determine the tool that you should...

  18. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...centrifugation (lenti) Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher...will help you decide which is right for you. Table 1: Mammalian versus insect cells for viral production... (about three to four days), this second round provides the added benefit of producing a stock P1 virus...

  19. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    Published
    Oct. 22, 2015, 2:30 p.m.
    ... fusions that retain their individual activities (1). Researchers also discovered the modular format of...systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown ...ubiquitin system has been devised (8). References 1. Casadaban M.J., Martinez-Arias A., Shapira S.K., ...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ...

  20. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want...are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1]...biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https://doi.org/10.1038/s41467-024-52871-9...promising tool for future structure-guided drug design and studies of protein ubiquitination and degradation...CRBNmidi plasmid here!   Kroupova, A. et al. (2024). Design of a Cereblon construct for crystallographic and...specifically for C. elegans (Cao et al., 2024). The team designed coding sequences for mScarlet3 and mScarlet-I3...
Showing: 261 - 280 of 587 results