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We narrowed to 522 results for: des.2

Showing: 41 - 60 of 522 results

  1. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    Published
    Oct. 20, 2022, 1:15 p.m.
    ...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...gRNAs. Photocleavable guides are generated by introduction of a single photocleavable 2-nitrobenzyl linker...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...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...Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101: Ribonucleoprotein ...

  2. Antibodies 101: Fab Fragments

    Type
    Blog Post
    Published
    Sept. 5, 2024, 1:15 p.m.
    ...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

  3. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    Published
    June 22, 2023, 1:15 p.m.
    ...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...that are built into how something functions, were designed for this purpose. One such control is dividing...

  4. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ... recombinase (Figure 2). Figure 2: A knockout targeting vector designed to insert and later remove...process, we’ll walk through an experiment designed to knock out exon 2 of a given gene.   Figure 1:...   Design your targeting construct. For recombination to occur in a cell, a minimum of 2 kb of sequence...multiple ways. The desired recombination event will remove NeoR only and leave exon 2 floxed, as seen in...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...often use the Cre/Lox system described earlier. In this case, you design your targeting vector such that...

  5. Great Results Start with Great Standard Curves

    Type
    Blog Post
    Published
    Aug. 15, 2024, 1:15 p.m.
    ... Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series that ranges...series ranging from 1:2 to 1:32. Each individual step of the series is consistent at 1:2. When preparing a.... An example of this is shown in Figure 2.   Figure 2: The concentration of a serially diluted... ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration of ...accuracy of the curve, avoid pipetting small volumes (< 2 µL) or volumes too large for a standard micropipette... 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 ...

  6. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    Published
    Dec. 29, 2020, 2:15 p.m.
    ...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...databases, DNA sequence manipulation, and primer design. Best wishes for the new year and your research...

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

    Type
    Blog Post
    Published
    July 18, 2017, 12:41 p.m.
    ...Rodrigo Arturo Rivera Solís, Universidad Autónoma de Yucatán, Mexico Dr. Rodrigo Arturo Rivera Solí... genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which he will co-express together with a plasmid ...Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded...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 ...as insecticidal proteins, the use of chemical pesticides may be avoided or decreased. Biosensors for herbicide...

  8. Hot Plasmids - October 2022

    Type
    Blog Post
    Published
    Oct. 6, 2022, 1:15 p.m.
    ...and 2), they found an indicator that is optimized for 2-photon imaging of voltages. This includes monitoring...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors show that...

  9. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    Published
    Feb. 27, 2018, 2:04 p.m.
    ...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...

  10. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    Published
    Jan. 16, 2025, 2:15 p.m.
    ...). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron, 92(2), 372...α2,3-/α2,6 N-linked sialic acids1 AAV receptor (AAVR)2   AAV2 Heparan sulfate proteoglycan (HSPG)3 ...3 AAV receptor (AAVR; not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5...AAV5 2,3-N-linked sialic acid4 AAV receptor (AAVR)2 Platelet-derived growth factor receptor (PDGFR...Heparan sulfate proteoglycan (HSPG)3 AAV receptor (AAVR)2 Epidermal growth factor receptor (EGFR)3 AAV8... AAV8 Laminin receptor (LR)3 AAV receptor (AAVR)2 Carboxypeptidase D (CPD), also called AAVR25   ... Terminal N-linked galactose3 AAV receptor (AAVR)2 Laminin receptor (LR)3 Putative integrin3 ...

  11. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    Published
    July 15, 2025, 1:15 p.m.
    ...short-read sequencing are highlighted in Figure 2. Figure 2: Overview of short-read sequencing technology...software such as SPAdes, Shovill, apc, and NOVOPlasty. The pipeline contains three de novo assemblers,...manually checks and assembles the raw data.  Addgene’s de novo assembly pipeline usually generates the correct...raw FASTQ reads are used for either guided or new de novo assembly. If a full anticipated sequence is ... doesn’t resolve the assembly issues, we can redo de novo assembly within Geneious.  While this may seem...steps that gave us our initial incorrect assembly, de novo assembly within Geneious gives us additional...

  12. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual.... When contamination is suspected, we proceed to de novo assembly of the unmapped reads, employing our...transduced cells whenever possible. This post will describe our workflow for viral genome sequencing (VGS)...

  13. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    .... Okita et al describes a protocol for generating iPSCs by plasmid transfection where 2 of 11 clones tested... cell reprogramming factors and wait for cells to de-differentiate. However it’s difficult, especially...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...

  14. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    Published
    Feb. 22, 2017, 3:30 p.m.
    ... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

  15. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    Published
    June 16, 2020, 1:25 p.m.
    ... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...other uses.   Cloning grade DNA Molecular cloning includes a set of techniques that are used to insert recombinant...

  16. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    Published
    March 30, 2017, 2:30 p.m.
    ...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of ...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...to testing their performance (2). Remember that although digital PCR is a great screening tool, it still...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...your genome edits, this paper provides detailed instructions on how to design such assays, in addition to...great deal of precision. This means digital PCR provides an absolute measure of target abundance, does ...

  17. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    Published
    Jan. 25, 2024, 2:15 p.m.
    ...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...online, searchable resource that provides summary information on Class 2 Cas enzymes. The information is...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry...Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry, Biophysics... is also documented. Experimental design When you are in the design phase of your experimental planning...is organized in wiki format and includes biochemical properties, previously established uses, and intended...relevant resources The homepage displays a graphic describing CasIDs and relevant properties of Cas enzymes...

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

    Type
    Blog Post
    Published
    Oct. 30, 2018, 1:04 p.m.
    ...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate into...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular...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...

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

    Type
    Blog Post
    Published
    April 13, 2017, 2:30 p.m.
    ...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally PHO8 ...

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

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction... Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers. The...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals... CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...this article, Quilt CTO Aneesh Karve, shows how to design experiments that work anywhere in the genome. Aneesh's...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...
Showing: 41 - 60 of 522 results