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We narrowed to 736 results for: RAN-1

Showing: 181 - 200 of 736 results

  1. CRISPR 101: Non-Homologous End Joining

    Type
    Blog Post
    ...842 . https://doi.org/10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, ...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous...

  2. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    ... process. Also refer to Figure 1 for a visual overview! Figure 1: General overview of the MoClo... multiple colonies after each transformation. This could be as few as 1–2 colonies if you’re an experienced...part from an Addgene MoClo kit, to construct a Level 1 vector with a promoter and terminator flanking the...acceptor (bottom) for CDS1 parts. (B) Example Level 1 assembly. The choice between different Level 0 vectors...properly. Do this sequentially for your Level 0, Level 1, and Level 2 assemblies using the plasmid sequence... after each transformation and/or use different E. coli strains with higher transformation efficiency....vector with two other independently regulated transcription units. Created with BioRender.com.   Planning...

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

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

  4. The Importance of High Titer for AAV Transductions

    Type
    Blog Post
    ...methods to improve AAV production.     References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high...be required. For example, retrograde AAV gets transported up the cell’s axon. While the mechanism for this...yet fully understood, we can imagine that this transport is probably not 100% efficient. So the few AAV... higher retrograde AAV titers for successful transduction. In another example, neuronal gene expression..., fewer channel proteins are required for the transduced cell to become light responsive than fluorescent...process of generating viral particles. Limit tube transfer - the fewer opportunities your viral particles...

  5. Improving Transduction Efficiency with Exosome AAVs

    Type
    Blog Post
    ...vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles ... are often found with cellular exosomes, small membrane vesicles or microvesicles (approx. 40 - 150 nm... diameter) that are secreted from the cellular membrane into the culture medium. AAV particles are either... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...to modify it to improve yields. For example, transfecting the producer cell line with an expression vector...proteins, nucleic acids) which possibly improve transduction properties. György and colleagues found that...that the association of AAV particles with membrane vesicles protects them from antibody recognition and...

  6. Click Biology: How to Click and Play to Explore Biology

    Type
    Blog Post
    ...the best performance in the most contexts (Figure 1). Together the features with optimal biological performance...described as Click Biology (Howarth, 2025). Figure 1: Modular assembly tools, alongside criteria for Click...tools can be found in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite ...coupling. Created with BioRender.com.   Table 1: A handful of Click Biology tools available at Addgene... intein; N-terminal CLm and C-terminal Aes gp41-1 Split inteins A small, fast split intein SpyTag003...of the coupling system include: Wide fusion-tolerance — Click Biology tags should be similarly useful...parts in the micromolar or nanomolar concentration range. Reactions to happen under lots of conditions ...

  7. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ...). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...serotypes 1, 8 and 9 share conserved mechanisms for anterograde and retrograde axonal transport. Hum Gene.... (2009). Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat ... Brain Res 406(1-2), 317-321. PubMed PMID: 2436717. Towne, C., Schneider, B.L., Kieran, D., Redmond, D.E.Jr...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...

  8. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous recombination...sequences of interest to cells with efficiencies of 1-40% after G418 selection (Kohli et al 2004, Kim et...with polylinkers for the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor...since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there are ...efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%. While this is comparable...gene inactivation in human cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ...

  9. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    ...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little as 30...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...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...

  10. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in ...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ...

  11. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below) ...complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA duplex. Image from...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...who worked on this publication:         References 1. Richardson, Christopher D., et al. "Enhancing homology-directed...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...

  12. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ... microscopy contrast technique in modern biology (1). When performed correctly, fluorescence microscopy...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...of modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...

  13. 500,000 Plasmids Shipped and Counting!

    Type
    Blog Post
    ...here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around...start out at our Cambridge office – previously at 1 Kendall Square, but newly relocated to 75 Sidney Street...Have a question? Email: [email protected] Call: +1 (617) 225-9000         ...Addgene's lab. We ship individual plasmids as transformed bacteria in stab culture format. Our lab team... and collaborations with institutions and tech transfer offices that facilitate MTAs. The great feedback...

  14. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico...material is right for their experiment.     Table 1: Most popular guides, protocols, and collections of...in 2023, from visitors in 218 countries. In Table 1, you can see our most popular educational resources... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid... package stays in transit for a certain number of days and tracking average transit time for each courier...

  15. Deep Dive: qPCR

    Type
    Blog Post
    ...Issue 19, 1 October 2000, Pages 3752–3761, https://doi.org/10.1093/nar/28.19.3752Wong M; Medrano J. Real-time...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...in primer Yes Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from...show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak, indicating...changes in ΔΔCT are represented by RQs between 0 and 1. Flipping your experimental and control values during...see this fold change as a positive difference (RQ>1), which may be easier to conceptualize. Just remember... in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_3, © Springer Science+Business Media New...

  16. Celebrating Outstanding CRISPR/Cas9 Achievements at the Dr. Paul Janssen Award Dinner

    Type
    Blog Post
    ...minutes, which was fun to watch. A simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at...to direct cleavage the result was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the...this type of experimentation to the eventual translation to the clinic and cures. There is no doubt that...

  17. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...Gate cloning, perform an exonuclease cleanup, transform, plate, and pick. In short, the Fragmid system...AAV pRDA_889 (ITRs from AAV2) Mammalian Transposon pRDA_757 (Piggybac; constitutive expression...   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy insulator sequences; white + selectable...

  18. From Lab Bench to Leadership: Transitioning into Management as a PhD

    Type
    Blog Post
    ...pitfalls? Here's what you need to know.   Figure 1: Management can seem a mystery at first! Image used...Understanding the transition While a PhD equips you with exceptional technical skills, transitioning into management...   Strategies for a successful transition To successfully transition into a managerial role, it's essential... most pivotal decisions many PhDs face when transitioning from academia to industry is whether to stay...career trajectories within the industry, but the transition wasn’t easy - I had to reinvent myself and redefine...d like to share below. Common pitfalls in the transition (what I wish I knew before experimenting on myself...being a great scientist doesn’t automatically translate to being an effective manager. Mismanaging (no...

  19. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition...

  20. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...proteins, which could disrupt your target epitope (Fig. 1). So, while organic solvents may improve antibody ...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created...
Showing: 181 - 200 of 736 results