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We narrowed to 704 results for: des.1

Showing: 181 - 200 of 704 results

  1. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...limits of oligonucleotides. These donors traditionally have larger homology arms in the 500 bp to 1 kb range...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...knock-in. Then choose which Cas enzyme to use and design a gRNA to where you would like to introduce the...sequence to introduce, which is done through the design and use of a donor DNA molecule. Your donor molecule...

  2. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    ...kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... produced Ethical concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation...

  3. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...

  4. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...  Figure 1: Using Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA...the target sequence is present in the pool of nucleotides, the nonspecific RNAse activity of Cas13a becomes...

  5. Some Like it Hot: Thermostable GeoCas9

    Type
    Blog Post
    ...Figure 1 for a summary of the experimental results. GeoCas9 RNP editing in mammalian cells Besides being...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...

  6. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    ...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...Over the past decades, neuroscientists have made great strides to map brain regions and allocate specific...for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...

  7. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...including yourself as the researcher.   Figure 1: Separation of retroviral packaging components onto...these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that...lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus...required whenever there’s a splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time...isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may...that are built into how something functions, were designed for this purpose. One such control is dividing...

  8. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ... Figure 1: In conventional gene targeting (A), you must first generate your desired mutation in mouse...much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR... drug induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid...CRISPR ribonucleoproteins.” Genome Biol. 18 (2017) (1):92. DOI: 10.1186/s13059-017-1220-4 PubMed PMID: 28511701... using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038/nprot.2017.153 PubMed PMID:...engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059-018-1400-x PubMed PMID: 29482575...using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To design a knock-in allele at a given...

  9. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...Fig. 1). So, while organic solvents may improve antibody binding for some targets, they can destroy it ...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...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...critical to the outcome of your IF experiment and deserve careful thought and attention.   * There are other...

  10. 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         ...information in our lab software, labeling stabs with barcodes as they go. Stabs are organized by shipment and...

  11. Plasmids 101: Secondary Nanobody Toolbox

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    Blog Post
    ...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...their production requires animal sacrifice and precludes genetic engineering of a tag or fusion with a ... with secondary nanobodies conjugated with the desired fluorophores. In figure 4B below, triple colocalization...review. If you just want to use the nanobodies described in Pleiner et al, the key steps for generating...

  12. Clear and Accessible Writing

    Type
    Blog Post
    ...writing Let’s use Figure 1 as our example. Remember, our goal is to use words to describe the image in a way...information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient...are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification ... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a... to our website through the use of alt text to describe images in online spaces. Alt text is used by screen... your alt text, use all of this information to describe the image helpfully. You and your readers are ...learned a lot from listening to others' alt text descriptions, and we’d love to hear yours as well.  More ...

  13. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

    Type
    Blog Post
    ...transcription activation system co-opted from yeast (1). It is a Drosophila geneticist’s main workhorse to...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z. Marka...references and web links from my first post and start designing your first fly experiment! Many thanks to our...PMID: 28704652. 6. V. Rodríguez, D. Didiano, C. Desplan, Power tools for gene expression and clonal analysis...

  14. Plasmids 101: Mammalian Vectors

    Type
    Blog Post
    ...premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells Complexes...expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T ...Concentation*** Blasticidin bsd  HeLa, NIH3T3, CHO, COS-1, 293HEK Inhibits termination step of translation ...mammalian plasmid components, it may be useful to describe the means of introducing genetic material (such...the plasmid in the same way. Selection methods, described below, are typically employed when creating stable...

  15. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ... Figure 1: Exon skipping strategies for Duchenne muscular dystrophy. gRNAs can be designed to flank ...X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular dystrophy...diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID: PMC3922311...);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360...dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and they must...

  16. Early Career Researcher Toolbox: Social Media for Scientists

    Type
    Blog Post
    ...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...use Twitter as a scientist. While this post is designed with beginners in mind, there’s some great reminders...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...

  17. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...for more details on how they work!) Figure 1: Base transitions are edits between the purine bases...R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%... the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked (RBMX...excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org/10.1038/s41467-021-21559-9...transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org/10.1038/s41587-020-0609-x...machine learning methods. Nature Communications, 12(1), 4902. https://doi.org/10.1038/s41467-021-25217-y...

  18. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    ..., rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would...IgG2a>IgG2b>IgG1 (Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies...antibody binding and therefore the signal strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal...raised in a different species.      Table 1: Isotype considerations when choosing antibodies ...Elsevier. https://doi.org/10.1016/s0091-679x(08)60255-1  Manning, C. F., Bundros, A. M., & Trimmer, J. S. ... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...

  19. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...journal publication describing the plasmids, or (for deposits submitted after July 1, 2025) after three... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...

  20. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...Humana Press, pp 133–148. https://doi.org/10.1007/978-1-62703-293-3_10 Walter J et al. (2020) Synthetic nanobodies...https://doi.org/10.1101/2020.04.16.045419.  Wrapp D, De Vlieger D, Corbett KS, Torres GM, Wang N, Van Breedam...Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly select for...strongly to your target protein. The Seeger lab designed 3 different mRNA sybody libraries for use in the... randomized with a defined set of different trinucleotides (corresponding to different amino acids) in...for binding to the target protein. The protocol describes going through two rounds of phage display to increase...
Showing: 181 - 200 of 704 results