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We narrowed to 916 results for: tro

Showing: 221 - 240 of 916 results

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

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    Blog Post
    ...would like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done...guide’s recognition sequence is destroyed via mutation. By introducing a mutation in either the PAM or...Identify where in the genome you would like to introduce your knock-in. Then choose which Cas enzyme to...region of a gene, make sure that the PAM edit you introduce is a silent mutation, so you don’t accidentally...to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove the ...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLIFE. (2014)...efficient homology-directed repair and termperature-controlled genome editing. Nat. Com., 8(2024), (2017). 10.1038...

  2. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...emission Before we design your first panel, we will introduce some background on the color spectrum and the ... photons are absorbed by matter, they promote electrons within atoms to a higher energy state. This process... called excitation. After a short period, the electrons revert to a lower energy state, whereby a photon...those of absorbed photons. When photons excite electrons of a fluorophore, they can do so at a range of...by the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few...absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure 2: Excitation... real-life advantage of a brighter fluorophore? Strong brightness makes it easier to distinguish a signal...

  3. New Viral Vectors - Fall 2024

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    Blog Post
    ...-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls Bryan Roth New...CN3905) AAV PHP.eB Controls Jonathan Ting New viral prep pAAV-CaMKIIa-EGFP AAV1 Controls Bryan Roth New ...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3044) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13038 - pAAV-AiE0743m...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3038) AAV PHP.eB Controls Jonathan Ting New viral prep AiP12408 - pAAV-AiE0600m-minBG-SYFP2...pAAV-AiE0600m-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2408) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13863 - pAAV-AiE0888m_C4...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3863) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13905 - pAAV-AiE0441h...New serotype pAAV-Ef1a-DIO mScarlet AAV5 Controls Karl Deisseroth New serotype pAAV_hSyn-PdCO-mScarlet-WPRE...

  4. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

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    Blog Post
    ...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels... to deliver a 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. Three different splice... and expression of the full-length transgene in vitro and in vivo (Allocca et al., 2008). Shortly after...levels were lower than expression of wild-type dystrophin in normal muscles, mice treated with the overlapping...achieved ~80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines... can be from one of the transgene’s endogenous introns or comprised of non-endogenous sequences, such ...Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular...

  5. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ...commercial isothermal assembly master mix. Introduction of the strong transcriptional terminator BBa_B1002 ...decide which path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression...called allelic exchange. Here, a mutated allele introduced from a conjugative plasmid replaces the chromosomal...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is also a... evolved specifically for toxicity would allow stronger counter-selection than one merely co-opted toward...rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is absolutely necessary, because...Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible promoter, though Khetrapal...

  6. Antibodies 101: Multiplex Immunofluorescence

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    Blog Post
    ...colors you like. Controls and validation Can’t forget the controls! The necessary control experiments will...from Addgene), or be prepared to run some extra controls and validate the antibody yourself. If you can... we’ll walk you through the necessary types of controls for a multiplex experiment.  Check for non-specific...using a different antibody.  Prepare a positive control Prepare and label a sample that expresses your ...naturally or by overexpression, for a positive control. You can also confirm the secondary works as expected...S., Diaz, M. F. P., & Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. In....2018488117 Additional Resources on the Addgene blog Introduction to Antibodies Which Fluorescence Microscopy ...

  7. Deep Dive: qPCR

    Type
    Blog Post
    ...If you're just getting started, check out our Introduction to PCR.) But if you're ready to go beyond the...tips on probe chemistries, primer optimization, controls, and analysis.  As a rule of thumb, you can assume...experimental parameters (neighbor stacking energies, loop entropy effects, cation concentrations and a temperature...side and a 5% negative error on the other will introduce unwanted variation into your data. Try different...protocol may have to be re-optimized.  While troublesome, multiplexing your reference gene set can save...the fold change of each sample compared to your control. But as we said previously, just because it’s used...assumes the amplification kinetics of the target and control genes are fairly equal, an assumption that is not...

  8. Common Injection Routes in Mice

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    Blog Post
    ...tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to ...restrainer or anesthesia Tail vein or retro-orbital 27–30 < 0.2 mL Beginner (retro-orbital) to intermediate (tail...2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital...vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used...needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful...decision Hopefully this post has provided a good introduction to common injection routes in mice! When considering...

  9. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide... in a bacterial strain introduced to the population as methods of introduction. Engineering the bee gut... of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky activation...systems differ on how exactly the CRISPRi locus is introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives...CRISPR system targets these genomic locations, and introduces double strand breaks, the bacteria are unable...This approach provides a more granular level of control where individual bases can be modified. Cytidine... that could be fused to dCas9 and efficiently introduce cytosine-to-thymine substitutions in target sequences...

  10. Getting to Know Expansion Microscopy

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    Blog Post
    ...super-resolution microscopy or electron microscopy. Since its introduction in 2015 by the Boyden Lab, ExM...expand evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the...2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment...much to be done about that.  Second, expansion introduces distortions. The goal in expansion is for your...variation, your samples can tear or expand unevenly, introducing distortions in your final images. It is possible...a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; ...resources on the Addgene blog Antibodies 101: Introduction to Immunofluorescence  Antibodies 101: Multiplex...

  11. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...protein. With this control set, you should see your target protein IP’d in the positive control sample but not... Basics of Western Immunoblotting.) For mass spectrometry, elute in a urea-containing lysis buffer. Alternatively...permanently altering the structure of the protein.  Controls As with any experiment, it is critical to include...include proper positive and negative controls for an IP. When possible, perform the IP in parallel with ...interest and a sample that does not. Common negative controls include knockout cell lines or tissues that do... not in the negative control, confirming that the capture antibody is functioning as expected. In order...perform parallel IPs with beads only and an isotype control antibody. Alternative applications Oftentimes, ...

  12. Plasmids 101: Broad Host Range Plasmids

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    Blog Post
    ...1998). Regulatory elements within the ori also control copy number and stability, which influence the ...which consist of a plasmid’s ori and all of its control elements, from broad host range plasmids can be...Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium...hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium methylicum...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E. carotovora...spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus ...

  13. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    ...type of edit you need to introduce highly specific? Perhaps you need to introduce a single nucleotide modification...McDade and significantly updated in 2022 by Susanna Stroik.  The advent of CRISPR/Cas9 has made it easier ...right for your unique experiment. Here, we will introduce you to the wide array of Cas9s (many available...Knock-out cells or animals are engineered by introducing Cas9-mediated frameshift mutations in the gene...interest. The genomic target for Cas9 and the introduction of this mutation can be any site early in the...and knockouts Some experiments may require the introduction of a mutation at a specific site e.g., deleting...Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and...

  14. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ...your cell of interest: AAV tropism and route of administration AAV tropism is defined by the viral capsid...FRT, with AAV vectors are common approaches to control transgene expression by restricting expression ...regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational regulatory ...Combinatorial strategies can also be used for retrograde tracing: visualizing neurons that received information...region. In the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression...viral vector related blog posts Learn more about retrograde AAVs Resources on Addgene.org Read our AAV ...guide Learn more about our viral service Find retrograde AAVs at Addgene ...

  15. Filming Science Videos in the Age of Social Distancing

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    Blog Post
    ...video making.   Cycle One: The Ironing Board Control Center With the pandemic in full swing and everyone...Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming Forward Design: ...me to set up a camera in a room, but still have control over focus, zoom, stop/start record, and allow ...focus system. Follow focus systems allow you to control the focus or zoom on a camera lens without having...door (connected via bluetooth to the camera, to control some basic functions, including starting and stopping... complete with wireless video receiver, camera control, and extra hand sanitizer.    Cycle 4: Venturing...mount a battery to it if necessary. I could still control the camera from a distance if I needed to be close...

  16. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells ... of your target site is. If you are trying to introduce a homozygous mutation, it’s helpful to know how...knockout cell line is desired, any mutation that introduces a frameshift in the coding region of interest... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions....   Validating knock-in mutations You’ve introduced a donor template along with your gRNA and Cas9...your desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or...restriction enzyme sites? Never fear! You can introduce a silent, passenger edit to serve as a proxy for...

  17. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

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    Blog Post
    ..., that strongly binds the glycoprotein, avidin or a similar protein, streptavidin. This strong interaction...antibody based affinity purification and mass spectrometry approaches. Recently however, scientists have...classic antibody based affinity purification/mass spectrometry methods.  BioID: a promiscuous biotin ligase...ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient proximity...of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring...exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID...Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and proximity labeling." Nature methods...

  18. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...-phosphorylated or ‘wild type’ control protein. The ‘wild type’ control can be made one of two ways. First... model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent...non-phosphorylated recombinant protein and perform in vitro kinase reactions. Often, however, researchers lack...or have difficulties with kinase specificity in vitro. The establishment of a system in which purified...-containing GFP that can be used as a positive control during optimization experiments. This version of... pSer incorporation can also done using mass spectrometry to identify a mass shift proportional to a single...expression plasmid. This is a particularly important control when creating a phosphoserine-incorporation mutant...

  19. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies such that the virus ... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Read on to learn how CRISPR can take on HIV-1 in vitro, and what obstacles must be overcome for clinical...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing with ZFNs and...infection period rather than the ART-induced viral control found in most HIV-1 patients. Obstacles for CRISPR...

  20. Summer SciComm: Science Storytelling

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    Blog Post
    ...creative commons license.    The introduction The introduction serves two purposes. First, it should... to have conflict. There are a lot of ways to introduce conflict into your work, but one of my favorite... two other things to complete your story: an introduction, and a resolution.   Fig. 2: The structure...ever thought about how X works?”, or even by introducing a person that the audience can relate to. It ...read (or listen or watch) further.  Second, the introduction needs to provide enough information so that ...thing applies for science storytelling. You can introduce your results right at the beginning of your communication...Addgene blog SSCS: Cognitive Load SSCS: Preprints Intro to our Summer SciComm Series  ...
Showing: 221 - 240 of 916 results