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We narrowed to 563 results for: cat.3

Showing: 461 - 480 of 563 results

  1. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    ...Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will promote depolarization...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...

  2. Ligation Independent Cloning

    Type
    Protocol
    ...reaction, meaning that T4 Pol will remove bases from the 3' end of the cut site until the first G is reached ...minimum of 18 bp of your template sequence. 5' and 3' primers will have different leader sequences, but... the same principle (homologous to the first G on 3'-5' strand from cut site). For simplicity, only the...salt concentrations in subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector...vector with T4 DNA polymerase to "chew back" the free 3' ends, following the manufacturer's instructions. ...treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... plasmid together through the transformation/replication process. LIC employs long overhangs to form a...

  3. Addgene-Seeding Labs plasmid grant round 3: microbial drug resistance and biofilms, antidiabetic plant components, visualizing brain injury - and a glowing robot fish

    Type
    Blog Post
    ...fields and geographical locations. In addition to providing reagents and educational resources for scientists...formation. The quest for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate...nephropathy, one of the major pathophysiological complications of diabetes mellitus. Recombinant human proteins... employing synthetic biology resources and microfabrication technologies to generate a self renewable ... in the river, take water samples at specific locations, measure fluorescent signals from the sensor, ...

  4. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you...enzyme on the 5' end and a different enzyme on the 3' end). This ensures that the insert will be added ...cloning (where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine....reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient, you...on the length of the DNA to get a proper ratio of 3 available insert ends for every available vector end...performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages...troubleshooting failed ligations. The following table indicates the various controls: Control Ligase Interpretation...

  6. Plasmids 101: Protein tags

    Type
    Blog Post
    ...tag than protein), 2-3 tags is increasingly common. Tandem affinity purification (TAP) once referred specifically...or DYKDDDK 1 Affinity and Purification Good for antibody-based purification; has inherent enterokinase...site GST Large Protein 26 Purification and Stability Good for purification with glutathione; protects...used for purification as elution requires low pH poly His HHHHHH  0.8 Affinity and Purification Very small...GKPIPNPLLGLDST   1.4 Affinity and Purification Good for antibody-based purification Combo and cleavage tags...multitude of uses including (but not limited to) purification, detection, solubilization, localization, or...fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...

  7. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...immunoprecipitation (enChIP)." Scientific reports 3 (2013): 3171. PubMed PMID: 24201379. PubMed Central...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...the initial report describing CRISPR-mediated purification of a specific genomic region, numerous adaptations...

  8. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...corresponding Excel sheet.3. Be specific  - When assigning a physical location to your samples, include.... Tracking samples - it's all about location, location, location There are many different types of samples...but we also have a dedicated and detail-oriented lab team that works carefully to catch mistakes. Each sample...sample a unique location in a specific freezer will help ensure that samples are easy to locate in freezers...also help us keep track of the tubes’ physical location in the lab. These barcodes additionally allow ...Addgene. Each sample at Addgene has four layers of location which can all be extrapolated from the 2D barcode...Plate Freezer Rack Freezer Tubes are assigned a location within a plate that can hold up to 96 tubes (see...

  9. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...addiction module" regulated by guanosine [corrected] 3', 5'-bispyrophosphate: a model for programmed bacterial...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...

  10. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...Magnetogenetic Ion Channels. Biophysical Journal, 116(3), 454–468. https://doi.org/10.1016/j.bpj.2019.01.003... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...changes in response to a ~25 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced..., in response to a ~10 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced...

  11. Protocol - How to Design Primers

    Type
    Protocol
    ...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template...proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches...stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward ...restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream in order...primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower...which creates primer dimers and disrupts the amplification process. When designing, if unsure about what...

  12. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...sample to a mastermix. At Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. .... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...detection of the product. As an added benefit, replicates are built into the technology. One well (containing...droplet contains the material required for a mini amplification reaction to take place.  When the PCR is complete...number of cycles. After additional rounds of amplification, your fluorescence amplitude will increase, ... use ddPCR for AAV titration, there are many applications for the technology.  Droplet digital PCR is ...

  13. Viral Production at Addgene

    Type
    Blog Post
    ...darker than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation shows...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images).     ...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...typically prepared using endotoxin-free plasmid purification kits.     Figure 4: Schematic of the...

  14. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872. Find plasmids...RESCUE in therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging...longer stretches of duplexed RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by...into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the construct... to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...construct for viral delivery. They found C-terminal truncations allowed the same or improved editing ability ...

  15. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...specifically to the blue triangle protein but not the other. 3) The beads are collected by centrifugation or a magnet...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

  16. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    ...–798. https://doi.org/10.1016/s1097-2765(02)00496-3 Kerppola TK (2006) Design and implementation of bimolecular...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...

  17. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    ...PMID: 25599551. PubMed Central PMCID: PMC4344395. 3. Lavis, Luke D., and Ronald T. Raines. "Bright ideas...Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules...phosphopantetheinyltransferase) – These enzymes catalyze the covalent attachment of a fluorophore ligand...labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores are often bulky and...the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent Protein Background Which ...

  18. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    ...Strong yeast expression promoter from glyceraldehyde 3-phosphage dehydrogenase Constitutive  Very strong,...resistance gene and the origin of replication are necessary to replicate plasmids and make sure the cells...length version is strong with high expression. Truncated promoters are constitutive with lower expression...metabolic operon Inducible by arabinose and repressed catabolite repression in the presence of glucose or by competitive...

  19. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ...bell or desiccator. Successful infiltration can often be seen by sinking of the tissue (see Figure 3). This...vincristine, are regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our ...expression method for analyzing gene function in Catharanthus roseus seedlings." Frontiers in plant science...

  20. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    ... sequence complementary to the mRNA sequence. The 3’ end of  the tRNA binds the amino acid that corresponds...folding and stability, change post-translational modification sites and even affect protein function (Mauro... AP, Bilen B, Zavolan M (2013) Analysis of CDS-located miRNA target sites suggests that they can effectively...synonymous codon usage bias, and its potential applications. Nucl Acids Res 15:1281–1295 . https://doi.org...
Showing: 461 - 480 of 563 results