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We narrowed to 587 results for: des.1

Showing: 1 - 20 of 587 results

  1. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    Published
    Oct. 11, 2018, 12:30 p.m.
    ... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture...representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ ...lacZ cassette, and illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological...over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases in the... 16 ºC) x 30 (100 μl) 78 90.7% 783 9,792 Table 1. Yields and fidelities for Golden Gate Assemblies ...

  2. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    ...marker. Looking at Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart...brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores...efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining...Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of a few commonly...Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores...blue laser excites at a wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation...Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation...

  3. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...dive in!      Figure 1: The technical decisions to make when designing a western blot.  Psst!...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...gel chemistry and/or specialty gel options. Table 1: Types of gels  Gel Type Protein sizes Running... Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...

  4. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ...Morgens et al. contains three types of guides: 1) targeting guides (blue), which target all protein-coding..., the toxicity of guides with off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets...) or short (17-18 bp) guides. When comparing the toxicity of guides that have 1-bp mismatch off-target... Figure 1: Summary of the advantages of the new approach to genome-wide CRISPR screen design.   Bassik... in both screens, with the growth screen having a 1% false discovery rate and identifying >88% of previously...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated than...Knockout Library are available from Addgene! References 1. Morgens, David W., et al. "Genome-scale measurement...

  5. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    Published
    Sept. 26, 2019, 12:50 p.m.
    ...~50% Table 1: Nanoblades editing efficiencies of primary cells. Comparing Nanoblades to Existing CRISPR...components into 293T cells. Figure 1: The key components of Nanoblades. Image from Mangeot et al., 2019....loaded with 1-4 different gRNAs. Viral envelope proteins To alter the cell tropism of Nanoblades, different... ~50-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved like...mouse-model generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle that’s based...) are expressed to pseudotype Nanoblades. The team found that Nanoblades pseudotyped with a mix of VSV-G...using Nanoblades? Plasmids are available from Addgene. Genome editing primary cells with Nanoblades Primary...

  6. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    Published
    June 1, 2023, 1:15 p.m.
    ...vectors. PLoS ONE, 9(8), 1–10. https://doi.org/10.1371/journal.pone.0106472 de Backer, M. W. A., Brans,...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...goals when designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, ...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License. ...

  7. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...cells will still appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity...gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA...cancer cell line in about 1 month, which I’d say is pretty amazing. Despite the shifting landscape of ...Materials Used 1. lentiCas9-Blast (Plasmid #52962) was a gift from Feng Zhang and is described in Improved...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then used to generate a monoclonal...rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9...

  8. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    Published
    June 17, 2025, 1:15 p.m.
    ...vectors. BMC Biotechnology, 6(1). https://doi.org/10.1186/1472-6750-6-1 O’Brien, J., Hayder, H., Zayed...endogenous miRNA machinery for processing (Figure 1). The shRNA sequence is transcribed to produce hairpin... recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and silencing...,"rgb":"rgb(0, 82, 169)","rgba":"rgba(0, 82, 169, 1)"},"button_font":{"size":19,"styles":{"font-family...:"rgb(255, 255, 255)","rgba":"rgba(255, 255, 255, 1)"}}{% endraw %}{% end_module_attribute %}{% module_attribute...297. https://doi.org/10.1016/s0092-8674(04)00045-5 De Rie, D., Abugessaisa, I., Alam, T., Arner, E., Arner...Gebhard, C., Goldowitz, D., Guhl, S., Ha, T. J., . . . De Hoon, M. J. L. (2017). An integrated expression atlas...

  9. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    Published
    Feb. 18, 2015, 3:09 p.m.
    ... nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara...harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration...from the University of Pennsylvania.     References: 1. Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions...11;342(6155):253-257. PubMed. 3. Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda T, et al. Characterization...and Bauer lab's related paper describes their methods for CRISPR design, cloning, and delivery, as well...

  10. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

    Type
    Blog Post
    Published
    Feb. 7, 2019, 2:16 p.m.
    ...This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD score) is...converted to a weight (which ranges between 0 and 1) so that they can be used in the rank equation above...converted to a weight (which ranges between 0 and 1) (shown in the graph below) so that it can be used...experiment in the program Design gRNAs by inputting the target regions Design donor RNA Design primers Review and...time for this project was spent selecting guides, designing primers, and running analyses. New team members...originals if desired. It works as a whole or in part. For example, you can analyze experiments designed outside...CrispyCrunch, or you can input pre-existing guides for primer design. The ecosystem of CRISPR tools Before...

  11. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the overarching...possible, do at least two full length practices (~1 h) and get feedback. It’s also helpful to practice...the knowledge gap? This includes the overview and the individual aims. Describe the question, the bare ...often hear, "I have an interview next week and it includes a chalk talk. What should I include in my chalk...may not actually help. Ultimately, if you cannot describe the likely outcome with some specifics, the search...cannot either. Therefore, you need to be able to describe experimental approaches in terms of the question...approach, anticipated outcomes, and preferably a description of the payoff if the aim is successful. Note ...

  12. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ...References Akhtar, W., De Jong, J., Pindyurin, A. V., Pagie, L., Meuleman, W., De Ridder, J., Berns, A....evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...this means we could design hundreds to thousands of different pegRNAs for each desired edit, it simply becomes...

  13. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    Published
    Nov. 7, 2017, 1:59 p.m.
    ...thanks to our guest blogger, Harshana S De Silva Feelixge. Harshana S De Silva Feelixge is a researcher whose... post was contributed by guest blogger Harshana S De Silva Feelixge. Gene therapy technologies hold ...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene...form, translating gene therapies from conceptual design to clinical trials involve identifying a therapeutic...

  14. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope... the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats (LTRs) that...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first...plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due to a few... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...lentivirus in the comments section below! References 1. In vivo gene delivery and stable transduction of ...

  15. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    Published
    July 15, 2025, 1:15 p.m.
    ...software such as SPAdes, Shovill, apc, and NOVOPlasty. The pipeline contains three de novo assemblers,...and recorded as a DNA strand is synthesized (Figure 1). Sanger sequencing requires four separate reactions...trace” file to determine the sequence. Figure 1: Overview of Sanger sequencing technology. Created...manually checks and assembles the raw data.  Addgene’s de novo assembly pipeline usually generates the correct...raw FASTQ reads are used for either guided or new de novo assembly. If a full anticipated sequence is ... doesn’t resolve the assembly issues, we can redo de novo assembly within Geneious.  While this may seem...steps that gave us our initial incorrect assembly, de novo assembly within Geneious gives us additional...

  16. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...allele. For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides, restricting...Vazquez F, Parts L, Boehm JS, Golub TR, Hahn WC, Root DE, Garnett MJ, Tsherniak A, Iorio F (2019) Agreement...770–788 . https://doi.org/10.1038/s41576-018-0059-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran ...j.stem.2014.04.020 Yang L, Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs... approaches, and provide updates on how best to design gRNAs. You can also find validated gRNAs for your...important to remember that the same is true when designing gRNAs for using CRISPR technology – the “best”...and sequence are important considerations for designing your gRNAs. For indels, it's not so important ...

  17. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

    Type
    Blog Post
    Published
    July 29, 2025, 1:15 p.m.
    ... Oligos Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1). Oligo 2: Add... Figure 1). Perform standard phosphorylation and annealing of the two oligos. Figure 1: Components... gene editing through de novo induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi....step-by-step guide for using a Dra2TnpB. Step 1: Design the 20-nt Guide Sequence Identify a TAM site ... promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene...protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20  Karmakar, S., Panda, D., Panda, S...1166–1187. https://doi.org/10.1038/s41477-021-00991-1  Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X.,...

  18. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...using a metric that we describe in the Materials and Methods section of our paper (1). Lastly, it is possible...Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene expression." Cell 159.4...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register...performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160...

  19. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    Published
    May 13, 2025, 1:15 p.m.
    ...and no viral progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral...2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https... cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers investigating...you might choose an IDLV over a lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors...Brugman, M. H., Pike-Overzet, K., Chatters, S. J., de Ridder, D., Gilmour, K. C., Adams, S., Thornhill,..., H. E. (1996). Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity...Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration in the human genome favors active genes...

  20. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    Published
    March 28, 2024, 1:15 p.m.
    ...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.4] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...specific integrins in cellular processes.   Figure 1: Integrins can shape into three conformations: (A)... 26 recombinant antibodies (rAbs) either produced de novo using yeast display technology or produced from...immunoprecipitation and immunohistochemistry. Table 1: IPI antibodies  Antibody Target Proteins Reactivity...Human IgG1 Rabbit Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG1 Rabbit Anti-Integrin...Human IgG2a Mouse Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG2a Mouse Anti-Integrin...
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