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We narrowed to 698 results for: des.1

Showing: 551 - 600 of 698 results

  2. Plasmids 101: Origin of Replication

    Type
    Blog Post
    ...produce as many as 700 copies per cell. Figure 1: A plasmid map showing the standard features of a ... A -C compatibility grouping is an arbitrary designation, and plasmids from the same incompatibility group...

  3. Advice for Moving Into Sales After Your Science Postdoc

    Type
    Blog Post
    ... After all I was only going to be interviewed for 1 hour so having a broad enough insight was going to...Coordinator’ came up at Abcam. Unlike other job descriptions for marketing positions posted on recruitment...

  4. Hot Plasmids: FRET-Based Biosensors

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    Blog Post
    ...variants. This resulted in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants...beginnings of the field. Those engineered sensors were designed to replace synthetic calcium dyes, which had been...

  6. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    ...can also rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth distribution” curve...next-generation sequencing (NGS). For gRNA libraries, you’ll design primers to create 200-300 bp sequencing products...

  7. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    ...loci in the genome and the guides are relatively unstable.   Figure 1: CRISPRainbow labeling. Two... the gRNA tetraloop and went through subsequent design iterations to increase stability and minimize misfolding...

  8. The Importance of High Titer for AAV Transductions

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    Blog Post
    ...methods to improve AAV production.     References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

  9. DAP arrays for multiplex gene editing

    Type
    Blog Post
    ... genes with a single viral vector.   Fig. 1: Schematic of multiplex gene editing with DAP array...The natural CRISPR locus of a bacteria host encodes multiple guide RNAs (gRNAs) on a single array to ...applications, we provide detailed manual and online design examples of using Addgene plasmids hCtRNA_FT (#186715...

  10. Hot Plasmids - January 2023

    Type
    Blog Post
    ...as an endogenous RNA imaging tool.   Fig. 1: Csm, shRNA, and Cas13 were compared head-to-head ...effects, both of happen with Cas13. CRISPR-Csm degrades both nuclear and cytoplasmic mRNA with 90-99% ...crystalizing the hfYFP structure, the group rationally designed GFP variants with similar capabilities. If you...

  11. Four Factors that Differentiate the Stem Cell Field

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    Blog Post
    ...regarding what makes this group of researchers special: 1. Stem cell scientists care deeply about ethics Ethics...speakers from top minds in the stem cell field have described the Yamanaka paper as "life changing". Dr. Yamanaka...affordable. 4. Stem cell scientists are generous Besides some of the scientists I've already mentioned, ...

  12. Antibodies 101: Flow Cytometry Controls

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    Blog Post
    ...settings with experimental samples. Figure 1: Example flow plots for a single color control. A)...familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as...controls like FMO or isotype controls, which we’ll describe a bit later. Single Colors Single color controls...

  13. Will You Be My Mentor? Finding and Asking for Mentoring Support

    Type
    Blog Post
    ...Networking 101-Building Relationships for Success”. Choose 1-2 tactics to try. One of my favorites is making the...mentoring questions I set out to answer. First, I will describe some of the many ways you can approach finding...Watch for my upcoming mentoring post in which I describe how to organize a successful peer mentoring circle...

  14. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    ...and CasPEDIA have got you covered!     Fig. 1 – CasPEDIA Cas ID display information for a given ... is also documented. Experimental design When you are in the design phase of your experimental planning...CasPEDIA is an online, searchable resource that provides summary information on Class 2 Cas enzymes. The...The information is organized in wiki format and includes biochemical properties, previously established...relevant resources The homepage displays a graphic describing CasIDs and relevant properties of Cas enzymes...What other RNA components are required? CasPEDIA provides information on crRNA and tracrRNA requirements... also show you how multiplexed arrays would be designed, if that’s your end goal. CasPEDIA also features...

  15. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

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    Blog Post
    ...into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct. Image from Wen ...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...constructs for the reporter to work and to make the desired genome edit:  A donor plasmid that contains a ...OH-T and doxycycline. A gRNA that targets your desired gene (whose sequence is also found in the reporter...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...integrated GFP reporter leaving you with just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene...DSB is repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame...

  16. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ...accessible to practically everyone.     Fig. 1: Open source hardware builds   Since I started...provide the construction plans or CAD (computer aided design) drawings of their devices, which makes it very...has allowed the community to regularly replicate designs worldwide. Distributing hardware via globally established...similar way to the biological protocol, and assembly designs based around the use of commercially available ...users outside the project to modify the raw and design files, allowing the device to take on a community-driven...of work from universities and research centers provides a potential conflict between traditional IP-based...

  17. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

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    Blog Post
    ...sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple... also high off-target activity, which may be undesirable in many applications. At the time of our first...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed...

  18. How to Keep a Lab Notebook for Bioinformatic Analyses

    Type
    Blog Post
    ...the output of the analysis performed in experiment 1. Use camelCase: In camelCase, each subsequent word...instance, your Desktop directory contains the folders and files that you see on your desktop. The location...immediately write a text document with a brief description of the directory contents, at a minimum. This...for a single use, save the script as originalName_descriptionOfEdit in the same directory where it was ...Your Next Experiment Find Our List of CRISPR gRNA Design Tools ...

  19. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    ...endogenous genes and it consists of three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A...first generation of CRISPR-Cas9 plasmids were not designed with gene activation in mind. “We had not managed...Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call CRISPR/Cas9...from the structure to guide and improve activator design,” she said. “Before it was a little like tapping...instead of at the protein’s C terminus. With that new design in place, Konermann immediately saw an order of...consistency and validation with >80% effectively enriched guides per gene hit, and 100% validation of the top 10...

  20. Summer SciComm: Preprints

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    Blog Post
    ...medRxiv.org) in the past few years.   Figure 1: Stand aside, CERN — it's a new age of accelerating...been common in fields like math and physics for decades (see: arXiv.org), but have been only adopted by...course, preprints don’t replace journals and other modes of scientific communication. Researchers often post...to receive deposits of plasmids that have been described in a preprint. In fact, over 1,500 plasmids in...Depositing plasmids that were used in a preprint provides all the same benefits as sharing those plasmids...resources on the Addgene blog Summer SciComm Series: Modes of Communication Intro to Our Summer SciComm Series...

  21. Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering

    Type
    Blog Post
    ... Effectors.” Cell (2018) pii: S0092-8674(18)30207-1. PubMed PMID: 29551272 Plasmids from this paper ...New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in...Like other Cas13 enzymes, the Cas13d orthologs described in these papers can independently process their... high targeting efficiency, both of which are described below. Yan et al.: WYL-domain-containing accessory...produced very similar cleavage products even when guides were tiled across a target RNA, indicating that...

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

    Type
    Blog Post
    ...Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces...INTEGRATE consists of four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade...Marraffini’s lab took advantage of this lethality to design the first synthetic bacterial CRISPR system in ...gRNA cassette for plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE...citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...editing efficiency, perhaps due to its condensed design. For both systems, custom spacers/gRNAs are easily...et al., 2013), the Qi lab employed a gRNA-based design more familiar to those using CRISPR in eukaryotes...

  23. Improving Transduction Efficiency with Exosome AAVs

    Type
    Blog Post
    ...vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles ...producer cells and purification protocols often describe a process that begins with cell lysis to release...

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

    Type
    Blog Post
    ...through the use of nanoparticles. [6].    References 1. Zhang B. 2021. “CRISPR/Cas gene therapy.” Journal...ability to influence editing efficiency. In 2022, despite the increasing effectiveness of CRISPR/Cas9, scientists...

  25. 5 Great Apps for Lab Life

    Type
    Blog Post
    ...instead conveniently provide them on your smart phone. 1. Lab Archives by LabArchives The single, most important...with NEB enzymes, the app is quite helpful and provides a list of directions including which buffers to...

  27. 3 Challenges in Plant Synthetic Biology

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    Blog Post
    ...challenges involved in plant synthetic biology. Challenge #1: Public perception of synthetic biology One of the...have been a model organism for plant research for decades, and the biochemical pathways involved in central...

  28. Antibodies 101: Isotypes

    Type
    Blog Post
    ...region: IgM, IgD, IgG, IgE, and IgA.    Figure 1: Diagram of an antibody labeled with Fc, Fab, heavy...molecules are recruited by the antibody to help destroy and remove a pathogen. Different isotypes also ...well as the genes that encode those proteins are designated by the lower-case Greek letters μ, δ, γ, ε, and...

  29. Viral Vectors 101: Viral Vector Elements

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    Blog Post
    ...guide outlines these components.     Fig. 1 - Viral vector components of retrovirus  Adeno-associated...promoter should flank this component as well. On both sides of this promoter-gene combo are two long terminal...Laboratory Press; 1997. Principles of Retroviral Vector Design. Available from: https://www.ncbi.nlm.nih.gov/books...

  30. GCE4All: Making Genetic Code Expansion Accessible

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    Blog Post
    ...non-canonical amino acid at the site of interest (figure 1). While the codon used is often one of the three stop...through a seminal paper from Peter Schultz’s lab describing a technique that allowed for intentional incorporation...reagents accessible to your lab. The solution GCE provides almost endless possibilities for researchers, ...

  31. 7 Tips to Secure a STEAM Internship This Summer

    Type
    Blog Post
    ...advantage this cycle, consider the advice below:  1. Eligibility check Scrutinize every opportunity prior... such as citizenship status, academic year, and grades that serve as automated filters of consideration...and cover letter revised by your network, which includes your friends, family members, and professional...

  32. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    ...give these stabilized promoters a try. References 1. Segall-Shapiro, Thomas H., Eduardo D. Sontag, and...of copy number (Segall-Shapiro et al., 2018). Designing stabilized promoters To keep gene expression stable...ideally be optimized to promote generation of the desired product while avoiding the accumulation of toxic...

  33. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    ...native peptide bond at the split site. Figure 1: The base editor is split into two plasmids and co-transfected...showing the potential for broad use. This kit is designed for use with acceptor vectors from the MoClo Toolkit...tracing Article contributed by Erin Sanders CreLite, designed by the Eisenhoffer lab, is a new tool that increases...

  34. Plasmids 101: Cre-lox

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    Blog Post
    ...techniques?  Let us know in the comments! References 1. Bacteriophage P1 site-specific recombination. I. ...using a resistance marker; however, it is often desirable to remove the marker after the initial selection... Combining this with some of the loxP methods described above, a genetic modification can be restricted...

  35. GPCRs: How Do They Work and How Do We Study Them?

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    Blog Post
    ...internalization of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. GPCRs in chemogenetics...new receptors were termed Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Roth, 2016...What do smell, taste and sight have in common, besides being one of the five senses? GPCRs or G-protein-coupled...photons or odorants, to elicit downstream signaling cascades.  So let’s break down the name "G-protein coupled...then induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand...

  36. What's New in CRISPR - November 2021

    Type
    Blog Post
    ... Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo RNA...different labs have recently published work that provides a solution to this problem. A miniature CRISPR-Cas...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...Nature Biotechnology paper showcases an improved design of the guide RNA that caps the 3’ end with a structured...that mismatch repair (MMR) interfered with the desired resolution of prime editing intermediates. Building...sequence. As with other prime editing tools, the desired edits are templated by the 3′ extension of the ...

  37. Giving gRNAs a Facelift - Synthetic and Beyond

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    Blog Post
    ...incorporated at the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101: Ribonucleoprotein ...

  38. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    ... an entirely new affinity reagent.    Fig. 1: A comparison of an antibody (left) to several alternative... as the OG (as the kids say) affinity reagents deserved to be celebrated, there are many other amazing...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...°C and can refold correctly after denaturing.  Designed ankyrin repeat proteins, or DARPins, are genetically...a wide variety of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells. They...

  39. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ...many of which are available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity...serotypes The names of AAVs contain two numerical designations, separated by a slash, that indicate the ITR...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged... an option (McCarty et al., 2001). This AAV is designed so that the single stranded DNA folds back on ...

  40. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    ...see how you use them in your research!  References 1.Tang, Jonathan C.Y., et al. “A nanobody-based system...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...stable inside cells. Nanobodies binding GFP were designed in 2009. Subsequent work showed that nanobodies...green-red Cre-lox systems. T-DDOGs can also be designed with other DNA-binding domains, including the ...

  41. Viral Vectors 101: Types of viruses

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    Blog Post
    ...efficiently with adenovirus and AAV.   Table 1: Types of viruses and their features Choosing ...about a specific virus? We have you covered with guides on lentivirus, retrovirus, adenovirus, and AAV....

  42. Antibodies 101: Secondary Antibodies

    Type
    Blog Post
    ...with each protein of interest.    Figure 1: The direct approach (left) versus the indirect approach...affinity, many indirect antibody protocols are designed to take place over multiple days. Increased run...

  45. 3D Printing Meets CRISPR Cas9

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    Blog Post
    ... – a mechanical device that could be used to bend 1/8th inch steel wire into a backbone structure of a...BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite protein in the...favorite protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D...off a small, family-run business – 3D Molecular Designs – to commercialize some of the best instructional...reaction to this model on his blog. Or, for a description of how effective this model was in explaining... up close and personal, visit the 3D Molecular Designs web site. Or, if you are visiting a local classroom...check out our hands-on teaching tools that are designed to make the molecular world real for students....

  46. dTAG - You're it!

    Type
    Blog Post
    ...cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids that... molecule degraders” recruit the proteasome to destroy targeted proteins. We previously developed small...locus-specific knock-in of FKBP12F36V. Detailed protocols describing the use of these plasmids are available here....Nabet et al., Nature Chemical Biology additionally provides detailed information on cloning procedures used...degradation, and lead author on the recent paper describing the dTAG technology. Follow him on twitter @behnamnabet... 29581585. This reference contains the full description of the dTAG system. The dTAG system was employed...Conditional Mouse Models CRISPR 101: Cas9 Nickase Design and Homology Directed Repair Plasmids for endogenous...

  47. Plasmids 101: 5 factors to help you choose the right cloning method

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    Blog Post
    ...your finished constructs at Addgene!). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified...amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. ...following cloning methodologies have been extensively described in other articles; so if you are interested in...homology. You might also consider FastCloning, first described by Chaokun Li and colleagues. Here you simply ...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...restriction enzymes. In Golden Gate cloning, the desired end construct does not contain the type IIS recognition...This feature biases the reaction in favor of the desired assembly to make the process close to 100% efficient...

  48. CRISPR in the Clinic

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    Blog Post
    ...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for CRISPR...introducing a desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One...potential for treatment is no longer an accurate descriptor; progress in clinical therapy is more fitting...One treatment currently in clinical trials includes Cas3 (the shredder member of the Cas family) as part...CRISPR therapies have made significant clinical strides since we first wrote this blog post. The future...

  49. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ... CRISPR guides) while a single 72h harvest is best for larger genes such as Cas9. See figure 1 for an ...newer generation lentiviral packaging systems were designed to prevent the formation of replication competent...use 3 plasmids; an envelope plasmid that usually encodes VSV-G, a packaging plasmid containing structural...the best titer possible. While this information provides a great starting point, it may not translate to...

  50. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    ... little signal detected along the neurite (Figure 1.) Moreover, the group was able to demonstrate that...soCoChR-expressing system. Taken together, Shemesh et. al. describe a novel system for the precise mapping of neuronal...the single-cell level. Various soCoChR plasmids described in this work including the soCoChR-GFP plasmid...information about spatial and temporal organization. The design use to detect PKA activity can be generalized to...choice for FRET assays where a dark acceptor is desired due to its very low quantum efficiency. ShadowY...
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