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We narrowed to 698 results for: des.1

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

    Type
    Blog Post
    ... 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. Distribution to Industry

    Type
    Collection
    ... your account. We will notify you by email within 1 business day regarding your account verification. ...Plasmid Gene/Insert PI Kits Kit name Type PI Description High Complexity Golden Gate Assembly Standards...Pooled Libraries Pooled Library name Type PI Description HR700_TP53 Exon Mutation Libraries CRISPR Thorsten...Parkinson's Disease; For target validation and drug design Antibodies Addgene distributes ready-to-use recombinant...materials and intellectual property rights, and provides liability protection for material providers. Addgene...

  3. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...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...

  4. Caltech Systemic Capsids

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    ... Caltech and can help guide your decision. Figure 1: Decision tree for choosing your AAV capsid. Your ...tdTomato Control Boyden 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Control Fishell 99130 pAAV-mDlx-NLS-mRuby2...at the bottom of this page. For more in depth descriptions of each serotype, check out Addgene's blog on...Browse Available PHP.eB AAV ID Name Promoter Description Category PI Controls 28306 pAAV-FLEX-tdTomato... Browse Available PHP.S AAV ID Name Promoter Description Category PI 28306 pAAV-FLEX-tdTomato CAG tdTomato...Browse Available PHP.V1 AAV ID Name Promoter Description Category PI 104052 pAAV-CAG-DIO-EYFP CAG EYFP...Browse Available MaCPNS1 AAV ID Name Promoter Description Category PI 37825 pAAV-CAG-GFP CAG GFP Control...

  5. Technical Design of a Western Blot

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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...

  7. New England Biolabs Cell-Imaging Plasmid Collection

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    Collection
    ...cell surface proteins. J Am Chem Soc. 127(37):12770-1. PMID:16159249...label of their choice according to the methods described by George et al. (2004). The ACP and SFP Synthases...respectively) may be expressed and purified as described previously in Stachelhaus et al. (1998) References...

  8. Antibody Production

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    Collection
    ...formulation buffer is phosphate-buffered saline containing 1 mM sodium azide. This buffer is not compatible for...protocols, titering methods, and quality control are described below. Information for a specific antibody can...

  9. CRISPR Plasmids - Prime Edit

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    Collection
    ...available as a substrate for another round. Figure 1: Overview of prime editing. Created with BioRender.com.... The longer pegRNA also encodes a primer binding site (PBS) and the desired edits on an RT template. ...post . Need help designing a pegRNA? Try the DeepPrime (Link opens in a new window) design tool from Hyongbum...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Prime editing is a “search...pegRNA). As with a typical gRNA, the pegRNA is designed with a spacer that binds to a specific genomic...3’ flap binds to the PBS of the pegRNA and the desired edit is incorporated into the DNA by reverse transcription...

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

    Type
    Blog Post
    ...~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...

  11. CRISPR Plasmids - Bacteria

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    Collection
    ...64325 3xFLAG-dCas9/p-bacteria 3xFLAG-dCas9 pLtetO-1 Fujii Efficient isolation of specific genomic regions... to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template. ID ...CRISPR plasmids that have been designed for use in bacteria. CRISPR...Bacteria The following CRISPR plasmids have been designed for use in bacteria. Cut Fully functional CRISPR/...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Base Edit Catalytically... can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator...gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter...

  12. TALEN Guide

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    Collection
    ...coming closer to this DNA targeting ideal. Figure 1: (A) Representation of a zinc finger (ZF) nuclease...components of the array recognize individual nucleotides. For over ten years, zinc finger (ZF) arrays ...that came out in the spring of 2011, the groups describe a set of customized plasmids that can be used ... a TALEN kit through Addgene. This kit, first described in Nature Biotechnology in August, uses a serial...in Nature Biotechnology early in 2011 and they described their kit for assembling TAL effector arrays in...effector and TALEN kits here . References Efficient design and assembly of custom TALEN and other TAL effector-based...

  13. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...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. ...

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

    Type
    Blog Post
    ...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...

  15. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ... 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...

  17. Viral Vectors

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    Collection
    ...material to specific cells in an organism. Figure 1. Using viral plasmids to generate virus. A) Viral ...material (termed the viral genome) that, in general, encodes for more viruses. When a virus infects a host cell.... Which Type of Virus Should I Use? This table provides a brief overview of the properties of different.... More detailed information can be found in the guides for each virus type, linked above. Virus Expression...Core . Viral Plasmid Biosafety Resources The NIH provides basic guidelines for working with various types...

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

    Type
    Blog Post
    ...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...

  19. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...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 ...

  20. CRISPR Plasmids - Plants

    Type
    Collection
    ...Hyg Chen 86196 pYPQ141-ZmUbi-RZ-As Maize ubiquitin 1 none As Cpf1 Qi 91715 pKEE401 yes, cut S. pyogenes...Protocols gRNA Design Tools CRISPR Blog Posts The following CRISPR plasmids have been designed for use in ... to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template. ID ...CRISPR expression plasmids that have been designed for use in plants. CRISPR... can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator...gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter...

  21. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    ...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...

  22. Viral Production

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    Collection
    ...to determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...

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

    Type
    Blog Post
    ...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...

  24. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...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 ...

  25. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...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 ...

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

    Type
    Blog Post
    ...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...

  27. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    ...-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...

  28. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...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...

  29. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    ...across scientific disciplines. Scientific Data, 8(1), 1–11. https://doi.org/10.1038/s41597-021-00981-0 Wright...excellent at infecting our target neurons (Figure 1). However, this one set of experiments didn’t provide...yet none of that data was published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8...392–400. https://doi.org/10.1038/s41434-018-0028-5 de Backer, M. W. A., Brans, M. A. D., Luijendijk, M....purification and formulation. Molecular Therapy, 12(1), 171–178. https://doi.org/10.1016/j.ymthe.2005.02.021...Stimulation. Frontiers in Human Neuroscience, 14(December), 1–12. https://doi.org/10.3389/fnhum.2020.578687 Additional...of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve our Addgene...

  30. Addgene-Seeding Labs Plasmid Grant: Accelerating Science Globally - 5 Scientists, 4 Countries & 3 Continents!

    Type
    Blog Post
    ... compounds with various antimicrobial activities (1). She has developed a fast endophyte screening system...Rodrigo Arturo Rivera Solís, Universidad Autónoma de Yucatán, Mexico Dr. Rodrigo Arturo Rivera Solí...us coordinate and promote the awards. References 1. Martinez-Klimova, Elena et al. “Endophytes as sources...antibiotic” Biochemical Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et ...renewable biotechnology products and biosensors for herbicides as well as developing yeast as source for dietary...technology to engineer plants that are able to destroy DNA viruses, similar to the anti-phage CRISPR effect...cloning kit) she intends to make yeast capable of desaturating fatty acids. Identification of Insecticidal ...

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

    Type
    Blog Post
    ...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...Cutting HIV-1 out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing...

  32. Neurodegeneration Research Collection

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    ...one of three different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of...brain. The cause of this neuron death is unknown. Despite the idiopathic (having no specific known cause)...scientific community at low cost, with rapid delivery. Includes antibodies, viral vectors, animal models, purified...

  33. Crowdfight, a Platform to Boost Scientific Collaboration During COVID-19 and Beyond

    Type
    Blog Post
    ...postdoctoral researcher at Instituto de Investigación Biomédica de Salamanca (IBSAL) and one of the coordinators...prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who believe...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the... illustrates the power of collaboration, which despite the competitiveness of cutting-edge research, is...physics. Even non-scientists, such as lawyers, designers and other professionals, joined the platform; ...

  34. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

    Type
    Blog Post
    ...for the wild-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were... tested at the time of this study. Figure 1: Neutralization levels against spike pseudovirus decreases...lowest levels of cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran..., Gosnell BI, Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A (2021) Escape of SARS-CoV-2 501Y.V2...

  35. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    ...regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186/1754-1611-8-7. PubMed.  Related Resources...targeting specific sequences of RNA is highly desirable for extending the complexity of genetic circuits...Zhao and colleagues, we now have just the tool…designer PUF proteins! A newly available PUF Assembly Kit...In a single step, researchers can now assemble designer PUF domains for RNA specificity engineering. “...proteins The tool she and her colleagues led by Zhao described and demonstrated in a report published in The ...translational repressor. In principle, however, designer PUF proteins can be fused to other components ...standard BsaI in place of the high fidelity version (despite what the original paper says). In their hands, ...

  36. An Introduction to Adenovirus

    Type
    Blog Post
    ...unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune...an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...adenoviral vector infected cells does not require de novo viral gene expression: Implications for gene...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval...viruses infecting humans (from seven species, designated as letters A–G) exist. The life cycle of AdVs...

  37. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    ... help!  General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When...  Color Base FP Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles...

  38. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    ...replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences...are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector contains a viral ...extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail. pCXWB-EBNA-1 lacks oriP and is unable...expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...pCXLE vectors listed below in Table 1 are available at Addgene. Table 1: List of pCXLE episomal plasmids,...obtained and the subsequent reprogramming success [1]. Fibroblasts need to be cultured and expanded in ...reprogramming factor cocktail, and viral protein EBNA-1. Methods for reprogramming hiPSCs from PBMCs While...

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

    Type
    Blog Post
    ...different plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and... Special thanks for Dr. Dan Stone and Harshana de Silva Feelixge at Fred Hutchinson Cancer Center for...about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, N., Hausl, M.... repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan...replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged into the...

  40. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

    Type
    Blog Post
    ...type of choice in vitro is known as reprogramming [1]. The process can be divided into two stages: Dedifferentiation...//www.linkedin.com/in/kmukherjeephd/.  References 1. Hochedlinger, K. and R. Jaenisch, Nuclear reprogramming...from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. PubMed PMID: 20621044. PubMed Central PMCID...induced pluripotent stem cells. Hepatology, 2010. 51(1): p. 297-305. PubMed PMID: 19998274. PubMed Central... pluripotent stem cells. Nat Biotechnol, 2014. 32(1): p. 84-91. PubMed PMID: 24291815. PubMed Central ...keratinocyte lineage. Methods Mol Biol, 2014. 1195: p. 1-12. PubMed PMID: 24510784. PubMed Central PMCID: PMC4096605...induced pluripotent stem cells. PLoS One, 2010. 5(1): p. e8763. PubMed PMID: 20098701. 31. Tang, Z.H.,...

  41. CRISPR References and Information

    Type
    Collection
    ..., 2.4 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9...CRISPR/Cas design (Link opens in a new window) Program for designing optimal gRNAs. Provides feedback on...window) This tool helps design (10 different prediction scores), clone (primer design), and evaluate gRNAs...Addgene Church gRNA design and cloning gRNA cloning vector PDF, 118 KB Church gRNA design and cloning for ...window) CRISPR/Cas9 reagent generation; gRNA design; HDR design; Deep sequencing Cas9: pMLM3613 dsDNA donor...CRISPR resources and references, including gRNA design tools, CRISPR FAQs, and community forums. CRISPR...protoSpaceJAM is an all-around platform for CRISPR knock-in design at scale. It uses an open-source algorithm to automate...

  42. COVID-19 Resources

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    Collection
    ...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV...SARS-CoV-2 Viral Pseudotyping Collection - This page includes a table of all spike expression plasmids in our... industry is continually growing. ID Plasmid Description Industry PI Return to top Other Coronavirus Plasmids... SARS-CoV, MERS, BAT-CoV Plasmids ID Plasmid Description Industry PI Due to export control regulations...plasmids related to these genes below. ID Plasmid Description Industry PI For more information on these genes...CRISPR-Cas13: Open-access SHERLOCK research protocols and design resources (Link opens in a new window) Find Zhang...COVID-19 research in the table below: ID Plasmid Description Industry PI Do you have a suggestion for plasmids...

  43. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...of an E. coli K-12 strain. F- λ- ilvG- rfb-50 rph-1 NEB Stable   For cloning into and storage of lentiviral...

  44. Great Results Start with Great Standard Curves

    Type
    Blog Post
    ...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for... the data points generated by replicate values provides useful information about the accuracy of the curve...If the unknown sample’s measurement is too low, redesign the standard curve dilution series with lower ...

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

    Type
    Blog Post
    ...three-step approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers...in combination with the techniques from Table 1, provides us with powerful GPS-like techniques to explore... chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...genomic coordinates into meaningful experiments. Table 1: An emerging family of "GPS for the genome" techniques...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...

  46. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing oligos that avoid activating...activating MMR: 1) use a strain of bacteria that has key MMR proteins knocked out or 2) specially design ssDNA ...  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds- and ss-DNA Uses in Recombineering...Considerations dsDNA Insertion or deletion of >20 nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase... ssDNA Point mutations or changing a few nucleotides 0.1-1% or 25-50% when activation of MMR is avoided...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...

  47. Celebrating the 2024 Nobel Prize in Chemistry

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    Blog Post
    ...protein design goals, both from the Baker Lab and others. The introduction of de novo designed proteins...protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize. Created with BioRender.com...concept that this kind of protein design is possible. Although Top7 was designed simply to be stable and novel...Hassabis and John Jumper! The Baker Lab focuses on designing entirely new proteins, improving our understanding...team was the first to develop and publish a human-designed protein that was not based on any existing protein...least that we’ve seen so far!). They started by designing a novel topology, then iteratively generated 3D...got closer and closer to achieving it. Once they designed the protein computationally, the researchers expressed...

  48. Revamp Your Lab Meetings With Creative Virtual Collaboration

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    Blog Post
    ...interaction fades away the longer we are on it – so it is important to take breaks. After 1 hour (or another...those ideas and build from those.   Figure 1: Virtual lab meeting? No problem! You can still creatively...salmon_phd References Frassl MA, Hamilton DP, Denfeld BA, de Eyto E, Hampton SE, Keller PS, Sharma S, Lewis ASL...in scientific research and in any workspace. Re-design lab meetings with a focus on discussion and brainstorming...pivotal in writing publications/grants too. As described by several works (Wuchty et al., 2007, Vicens ...another designated time) of meeting, someone could raise their hands or signal the approach of a brain...prepares a coffee, or simply moves away from the desk to switch off a few minutes. The host could also...

  49. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific promoter or enhancer...

  50. Delivery Methods for Generating iPSCs

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    Blog Post
    ...) element and the cis-acting EBNA-1 from Epstein-Barr virus. EBNA-1 binds oriP and allows for replication... cell reprogramming factors and wait for cells to de-differentiate. However it’s difficult, especially...Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods for Generating ...-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These plasmids carry the origin...method Most iPSC research falls into two categories: 1) studies focused on better understanding the mechanisms...iPSCs experiences in the comments below! References  1. Sano, Masayuki, Minoru Iijima, Manami Ohtaka, and...
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