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We narrowed to 993 results for: Des

Showing: 141 - 160 of 993 results

  1. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we were very excited...introduced. Although we were able to introduce the desired mutations in genes associated with dementia, such...CRISPR from re-cutting the target sequence once the desired edit has been introduced. We found that blocking...only saw incorporation of the blocking mutation. Despite the fact that both the blocking mutation and our...thinking further about the distance relationship described above, we realized that, in addition to increasing...efficiency a guide RNA targeting a cut <10 bp from the desired mutation should be used. For a heterozygous event...

  2. Brzezinski Lab CRISPR Collection

    Type
    Collection
    ...can be designed using Benchling or using similar design tools and ordered as standard desalted oligonucleotides...phosphorylated oligonucleotides containing the guide target sequence: Up to two guides can be expressed...that utilize Golden Gate cloning to insert the desired guide RNA sequence by digestion of the backbone... #63798) Plasmids from Collection ID Plasmid Description PI Publications Kaufman, M. L., Goodson, N. B...

  3. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...different ways. We describe a number of issues with some of the experimental designs used to make these...of Amsterdam. The previous post in this series described a practical approach to selecting a bright fluorescent... protein. Photobleaching is the irreversible destruction of a fluorophore under the influence of light...photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable...photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent...large number of emitted photons before they are destroyed. Factors that affect photostability The photobleaching...fluorescence, which, in its simplest form, can be described by a mono-exponential decay (analogous to radioactive...

  4. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...need for PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site...accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library containing several... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted... a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple target... not work with C. elegans, several groups have designed modular toolkits to assist with tagging genomic...

  5. Cre-Lox and Other Site-Specific Recombinases

    Type
    Collection
    ...efficiency, selecting desired clones, and making transgenic mice. ID Plasmid Description Gene/Insert Vector...light”): Cre Recombinase Plasmids ID Plasmid Description Vector Type Promoter PI Additional Addgene resources...Return to top Flp Recombinase Plasmids ID Plasmid Description Vector Type Promoter PI Additional Addgene resources...Return to top Dre Recombinase Plasmids ID Plasmid Description Vector Type Promoter PI Additional Addgene resources...Site-Specific Recombinase Plasmids ID Plasmid Description Gene/Insert Vector Type Promoter PI Return to...target site (lox, FRT, rox, etc.) ID Plasmid Description Vector Type PI Additional Addgene resources Use...Pooled Libraries Pooled Library Name Type PI Description Mouse Tumor Suppressor Gene CRISPR Knockout Library...

  6. Evolution of Lab Techniques

    Type
    Blog Post
    ...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands of all...fluorescence activity as fluorescently labeled nucleotides are incorporated, a method called pyrosequencing...nanopore-based DNA sequencing which reads the nucleotides of a DNA strand as it slips through a tiny pore... the different electrochemical properties of nucleotides (8). Compared to older technologies nanopore-...

  7. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    ...Have you ever designed a CRISPR guide RNA and wondered why it is limited to only 20 bases, or why it’...understand the parameters of CRISPR experimental design. Let’s dive into some structural biology! Major... lobes, REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic domains, as well as...as the CTD (also known as the PI), while REC includes most of the bridge helix. Created with BioRender.com...Fact! Even though the Cas9:RNA complex is well described in the closed conformation, recent electron microscopy... does not include stem loop 3, and guide RNA nucleotides 11-20 are not resolved due to structural disorder...2017). Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing...

  8. Zebrafish Plasmid Collection

    Type
    Collection
    ... Large collections or kits are described below . ID Plasmid Description Gene/Insert PI Return to top Highlights...rerio ), named for the horizontal stripes on the sides of the adult body, is a tropical freshwater fish...contain tools for use in zebrafish research. This includes plasmids for genome editing, empty backbones, ...Periklis Pantazis Lab Multibow: digital spectral barcodes for cell tracing - Sean Megason Lab Zebrabow -...Du Lab. A set of pCS2+ backbone-based Gateway destination vectors (pGCS), bearing either amino- or carboxyl-terminal...promoters, six fluorophores, and empty vectors designed to streamline the production of transgenic zebrafish...zebrafish-specific gene expression database that provides information about when genes are active in developing...

  9. Deep Dive: qPCR

    Type
    Blog Post
    ...from Wong, 2018   Primers Design of primers and probes Primer design, of course, could easily be its...useful primer design resources in the References and Resource section. Validation Once designed, your primer.... For example, chemistries that rely on hairpin design of probes/primers often offer higher allelic specificity...Taqman) Low; can be reduced with a double-quencher design Yes, one Yes Yes Agarose gel or uMelt analysis...Reference Genes  Choosing a set of reference genes deserves more attention than it usually receives. Different... multiple reference genes whenever possible (Vandesompele, 2002), and it's possible to multiplex your ...documented in the literature, a geNorm study (Vandesompele, 2002) will help you select the appropriate ...

  10. COVID-19 Resources

    Type
    Collection
    ...scientists. Antibodies ID Recombinant Antibody Description PI 211756 Anti-SARS-CoV-2 Nucleocapsid Protein...secretion, and codon-optimized versions designed for your expression system. Some of the following...Plasmid Distribution to Industry page. ID Plasmid Description Industry PI Additional Addgene resources SARS-CoV...SARS-CoV-2 Viral Pseudotyping Collection – This page includes a table of all spike expression plasmids in our... SARS-CoV, MERS, BAT-CoV Plasmids ID Plasmid Description Industry PI Due to export control regulations...plasmids related to these genes below. ID Plasmid Description Industry PI Additional Addgene resources ACE2...Neutralizing nanobody plasmids And more! ID Plasmid Description Industry PI Addgene blog Finding nucleic acids...

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

    Type
    Blog Post
    ...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...Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG...anyone can find enhancers with Quilt. Seek and destroy enhancers with CRISPR Armed with the genome math...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers. The...attenuated retrovirus in the same family as HIV. By design, our lentivirii are genetically programmed to CRISPR...Lentivirus from Addgene If you’re interested in designing your own CRISPR screens for enhancers, check out...

  12. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...their specificity or from the thymus to the lymph nodes. These methods gave researchers much more information...expressing cells. For example, by harvesting lymph nodes at different times after antigen challenge, scientists... cellular dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists...extravasate from the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon...interactions in diverse tissues, including lymph nodes, brain, liver, gut and skin. Two-photon intravital... 2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et...dynamics within the germinal center of murine lymph nodes. However, one must pay attention to time scale when...

  13. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...many MoClo kits are designed with specific model organisms in mind, others are designed for flexibility. ...Synthetic biology in bacteria MoClo is commonly used to design TUs and multi-TU constructs for synthetic biology..., biofuels, and natural products without the undesirable side products generated during chemical synthesis.... phaffii, then the MoClo YTK Kit specifically designed for S. cerevisiae is at your service (Lee et al...chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within... are endless: genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization.... The Fragmid kit described above works in both mammalian systems and Drosophila. Similarly, the Multi-Kingdom...

  14. Plasmids 101: Shuttle Vectors

    Type
    Blog Post
    ...affects all kingdoms of life. Of course, that includes you. Besides the risk to the researcher, Zeocin has been...most yeast vectors and many mammalian vectors are designed to shuttle between species. Figure 1: Shuttle...host’s replication machinery. A shuttle vector designed for unrelated bacteria should include oris for...target species. Fun fact! Plant vectors are usually designed to shuttle between E. coli and Agrobacterium tumefaciens...protects against any of them. But different aminoglycosides work on different host species: kanamycin targets...media that supplies the key nutrient. The vector provides the missing synthesis machinery, and the cells...image for individual analysis. A shuttle vector designed for this approach in a mammalian cell experiment...

  15. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)... large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). This...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-... selected the immediate-early gene Bdnf, which encodes brain-derived neurotrophic factor (BDNF). Bdnf ...High-throughput screening strategies Although the tools described above make CRISPRa more accessible to neuroscientists...

  16. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ...reproducibility than others. Unfortunately, most ELNs are designed as closed ecosystems and act as data silos because...RSpace ELN supports reproducibility RSpace was designed in response to requirements that emerged from ...used in research, and data repositories. Below I describe how each of these kinds of connectivity facilitates... in institutional and lab servers specifically designed to hold large amounts of data. Although it may... use of other tools they use in research. This includes chat apps like Slack, Microsoft Teams, and Google...individual documents or more complex datasets into the desired repository. They also support entry of required...deposits will be made. The Dataverse integration is described in this video and the Figshare integration in ...

  17. Plasmids 101: Positive and Negative Selection for Plasmid Cloning

    Type
    Blog Post
    ...property by including ccdB on its destination plasmids. In the destination plasmid, the ccdB gene is flanked... rest do not grow. While there are many ways to design your positive selection strategy, here are a few...straightforward in cases where the inserted DNA includes an antibiotic resistance marker. After transformation...grow. However, often times, the plasmid backbone includes an antibiotic resistance cassette and you aren...backbone contains your gene of interest. Auxotrophy Besides antibiotic selection, you can select for a successful...prototrophy come into play during cloning if you’ve designed your experiment using an auxotrophic strain of...sites. To introduce a gene of interest into the destination plasmid, recombination sites must first be added...

  18. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    ...Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides for binding affinity...of Spinach. In doing so, they identified the nucleotides that contribute to the instability of Spinach...similar levels of fluorescence when bound to DFHBI despite being approximately half the length (Warner et ...also smaller than any form of Spinach - only 49 nucleotides in length - and is better able to maintain its... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...Broccoli, and share similar properties to Broccoli despite binding to a different small molecule. Notably,...

  19. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the ...mutated clones. Forward and reverse primers are designed to be complementary, but each primer may extend...Polymerase To ensure that you don’t introduce undesired mutations through the PCR process, you need a ...The resistance marker from the parental plasmid provides a mean for selecting for transformants which have...mutagenized plasmids Once constructs containing the desired modification have been identified (and confirmed...

  20. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ... Addgene plasmids by gene here), designing primers with your desired mutation, performing PCR to generate...multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA...purification steps and end-repair steps with well-designed fragment overlap sections, while still creating... guide you through both experimental and primer design based on fragment sequences and the polymerase ...enzymes are adjusted. Appropriate fragment and primer design results in covalently joined, scarless DNA – perfect...by assembling multiple fragments containing the desired mutations. This same process can be used simultaneously...oligo that contains a region of homology and any desired edits to the target region. Assembled plasmids ...
Showing: 141 - 160 of 993 results