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We narrowed to 958 results for: Tes

Showing: 361 - 380 of 958 results

  1. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ... it is standard practice to compare degradation rates between samples treated with or without an autophagy...ATG) in yeast. Atg8p (“p” stands for protein) associates with autophagosomal membranes, so tagging it ...monitor autophagy, but under some conditions it aggregates in an autophagy-independent manner. Additionally...induce autophagy, red but not green fluorescence accumulates in the vacuole. See Figure 2 for an example of...Autophagy. Methods in Enzymology Autophagy: Lower Eukaryotes and Non-Mammalian Systems, Part A, 33-42. doi...

  2. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    ...A4 and A8-A10. The researchers selected 8 of the tested ABE8 constructs (ABE8.8-m/d, ABE8.13-m/d, ABE8.17...2 guanines). This limits the use of ABEs as DNA sites that do not contain a suitable PAM cannot be edited...for primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... gRNA used (Gaudelli et al., 2017). The authors tested ABE8.17-m and found that one base change mutation...overall improved base editing capacity, even at sites that used to be difficult to target. In particular...

  3. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...review analyzing and explaining the antibody crisis states that up to 50% of all commercially available antibodies...to more complex brain systems such as non-human primates and humans. Since we need larger-volume antibodies...extensive library of monoclonal antibodies; Addgene collaborates with these companies to create and provide general...includes accuracy, strength, and reproducibility. We test the antibodies on human and mouse tissue and compare... distinct imaged pattern, and exposure times. To test the antibody’s reproducibility, each antibody is...

  4. Mouse Modeling, Part 2: Breeding and Crossing Mice

    Type
    Blog Post
    ...They will come in handy here! Crossing your F1 heterozygotes to each other will generate approximately 1 ...wild type at one or both of the alleles, or heterozygotes, are not a waste. Best practice is to use littermate...transgenic strain, the promoter being used should be tested in cell lines to determine how it impacts the expression...chromosomes recombine during meiosis, creating gametes that contain a mix of genetic material from each...recombination at the floxed site, your control should be littermates that express Cre but not loxP. With this two-...

  5. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ... to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection... systems. What’s the error rate? The Doench lab tested the Fragmid system by sequencing 60 individual ... assemblies created over five months. When they tested those clones (in duplicate), 93% had the correct...your backbone and genes contain only the Bbsl cut sites needed for the Fragmid cuts, clone in the appropriate...References McGee, A. V., Liu, Y. V., Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger...

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

    Type
    Blog Post
    ...functions due to mutations accumulated over time? Undergraduates can help to answer this question! The Pathways...can be relatively confident that the gene from the test species has a similar function to the gene in the...enzymatic steps (5). In their experiments, students test whether or not proteins from the fission yeast, ...Excel: Producing One Million Additional College Graduates with Degrees in Science, Technology, Engineering...PMCID: PMC4012490. 4. Hoffman, C.S., Wood, V., & Fantes, P.A. An ancient yeast for young geneticists: A...

  7. CRISPR in the Clinic

    Type
    Blog Post
    ...characterized by misfolding of the TTR protein, which contributes to the disease phenotype. Intellia initiated ...Biosciences is currently running this trial, with more updates expected in the coming months/years. The CRISPR-HIV... (Hussein et al). It employs Cas9 to cut at two sites within the viral genome to excise a large portion...disease, inflammatory diseases, blindness, and diabetes. Directions and obstacles for future CRISPR therapies... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

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

    Type
    Blog Post
    ...it ranks guides by CFD score, and it mutates HDR donor templates to avoid re-cutting when needed. It gives...each gene, we needed to design guide RNAs, donor templates and sequencing primers for quality control. At...sophisticated features of CrispyCrunch is how it mutates guide sequences to prevent re-cutting of the sequence... the above are options, the algorithm silently mutates codons in the guide sequence, one by one, from ...

  9. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...surprise and delight,” the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight...and Maria were skeptical. The team was careful to test the possibility that this might just be an artifact...area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV shows...tools.  Embracing science in the open The York Lab operates with a strong commitment to exploration and collaboration...self-publishing directly on his website (and often posting updates on social media) and freely sharing code, raw data...

  10. Viral Vectors 101: Voltage Indicators

    Type
    Blog Post
    ...mechanism of their voltage sensing component, which separates them into two distinct categories detailed below...phosphatases from sea squirts. This design structurally separates the two components of the indicator, which can...was dipicrylamine, a lipophilic compound that distributes itself within the plasma membrane as well. Another...particularly physiologically relevant in neurons, myocytes, and endocrine cells. It follows that the specific...component (i.e., when the dye is lipophilic and integrates into the membrane), the voltage sensing component...

  11. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    ...quality control, but does not perform functional tests. The newest genome engineer sharing the stage with...be less desirable if one has to target multiple sites in a single experiment. Also, the NGG PAM sequence...nearly 400 times. Researchers have been excitedly testing NgAgo's genome editing applications. So, how does...conducted a survey and asked researchers if they had tested and compared NgAgo head-to-head with CRISPR-Cas9...could not achieve indels with the NgAgo system indicates that it is very tricky to use and fussy to optimize...

  12. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ... plasmid with multiple, individual expression cassettes, having the genes expressed from the same cassette...described below. IRES Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single...originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic ... start with a plasmid that has multiple cloning sites flanking the multicistronic element(s), or you could...efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim...

  13. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    ...biological questions. The two most common uses are: 1) to test the expression level in a specific system (by measuring...occurs when energy transfer from one FP (e.g. CFP) excites the fluorescence of another FP (e.g. YFP). FRET...create the chromophore. This can be from a few minutes after it is translated to a few hours. For example...However, for most FPs it is a few seconds to a few minutes. Photostability can be affected by experimental...greenfluorescentblog.wordpress.com.   More Helpful Websites & Resources: FP guide at Addgene Interactive ...

  14. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...including: how closely homologous the off-target sites are compared to the on-target site, the specific...immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM sequences should...HR template vector design guidelines? A7: A few notes below are considerations for designing HR donor....with PX330 plasmid, clone in your target guides, test run them, and then select the best guide to co-transfect...site in the donor. For example, choosing target sites that span the knock-in gene. For making mutations...As a successful insertion will destroy the BbsI sites, a double digest should discriminate between positive...

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

    Type
    Blog Post
    ...cell types. Variance can be caused by cell cycle rates; rapidly diving cells will have higher HR frequencies...viability of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle...carefully (Lin et al., eLife)! Use a Cas that facilitates HR What if promoting HR was as simple as swapping...It can be! Cas12a is a Cas family member which generates sticky ends upon cutting. Cas12a may promote HDR..., Rouet, R., Vejnar, C. E., et al. CRISPR-Cpf1 mediates efficient homology-directed repair and termperature-controlled...

  16. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...insert-specific sequence. This is a “yes or no” kind of test, with a positive clone amplifying a product and ...primers. These primers are designed to anneal to sites that flank the insert site. A positive clone will...depends on your preferences. Either way, make sure to test your colony PCR primers before using them to screen...Pros Cons Insert-Specific Simple "yes or no" test results Doesn't tell you the orientation of ...of sterile water.  Pick 3-10 colonies in total to test, depending on the number of background colonies ...

  17. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital...Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. Targeted...oligonucleotides called morpholinos. When injected into zygotes, morpholinos complementary to a target mRNA inhibit...help elucidate the gene networks that transform zygotes into adults and explain how signaling pathways ...study human illnesses such as blood disorders, diabetes, cancer, and Alzheimer’s disease. Naturally, the...

  18. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ...quickly screen and investigate transcription factor candidates and the regulation of genes in the biosynthetic...save you a lot of time. Don't re-invent the wheel. Test different leaf ages Usually younger leaves work ...interest. Find all plasmids from the Lee-Parsons lab. Test different leaf infiltration methods Co-cultivation...transcripts in the plant but not from Agrobacterium. Test different Agrobacterium strains We used the disarmed... This allows you to quickly assemble vectors and test different ideas. We are a huge fan of the Golden...

  19. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...nil base editing efficiency at some genomic sites. Some sites may be inaccessible to base editors probably...cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting only a single strand...indel formation. After binding with DNA, the CBE deaminates a target cytosine (C) into a uracil (U) base....Gaudelli et al., 2017). Our vectors have two Bsa1 sites to clone the single guide RNA (protospacer+scaffold...

  20. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...country.   Figure 1: Number of countries Addgene distributes to per year, 2005-2023. We identified and offered... we contracted with so we can always offer the fastest shipping. However, there were issues that persisted... country are represented in blue; darker blue indicates more views from that country.   While written ... country are represented in blue; darker blue indicates more views from that country. Stats and figures...resources shared via social media, on collaborators’ websites, or as a recommendation in a Reddit thread crowdsourcing...
Showing: 361 - 380 of 958 results