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We narrowed to 801 results for: Ran

Showing: 281 - 300 of 801 results

  1. Trading Spaces - Visiting Scientist Edition

    Type
    Blog Post
    Published
    Aug. 22, 2023, 1 p.m.
    ...remote collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork...to explore this role, how to seek out such an arrangement, and how to operate once in that position. What... plan as well. Making logistical and funding arrangements Visiting scientist positions take commitment...structured visiting program, you will have to make arrangements on your own. This will include, but is not limited... limited to temporary housing, transportation, living expenses, and necessary reagents. Your primary lab...these expenses, or you may need to seek out travel grants available from third parties or your home institute...generate data that will contribute to a publication? A grant? A patent? It is never too early to discuss authorship...

  2. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    Published
    April 5, 2018, 12:42 p.m.
    ... Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have been shown...perform in vitro transcription, and use the IVT product as a template for reverse transcription to create your...order to insert two loxP sites. Since two double-strand breaks are made, it is common to see deletions ...traditionally been difficult to produce long single-stranded DNA, but Miura et al. (2015) developed the ivTRT...structure. Today, multiple companies offer long single-stranded DNA production services or kits, including the...microinjection and electroporation methods using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To ...used guides targeting the same or complementary strands, as well as guides facing towards or away from ...

  3. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...the co-transduction by two viruses would lower editing efficiency, the split editors transduced at levels...capacity of 4-5 kb limits the delivery of larger transgenes, such as base editors which have a gene size ...base editor is split into two plasmids and co-transfected into cells for reconstitution. Image from Levy...-opsin GEVIs. With an increase in the neuron’s membrane potential, Ace absorbance increases and results...promoters, CRISPRi parts, selectable markers, transformation acceptor vectors, and sequences for genomic...plasmids using the Cyanogate Kit and subsequent transformation into cyanobacteria. Image from Vasudevan et... evaluated CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this system...

  4. Announcing the Winners of the 2021 Michael Davidson and Roger Tsien Commemorative Conference Awards

    Type
    Blog Post
    Published
    Aug. 5, 2021, 12:15 p.m.
    ... from Abhi Aggarwal.   Brandán Pedre Pérez: Redox biosensors Brandán Pedre Pérez is a postdoc at... enable multiplexed imaging of neurotransmitter, calcium, and membrane voltage,” says Tian. Figure...Congratulations to the five winners! Abhi Aggarwal Brandán Pedre Pérez Elizabeth Hubbard Haoyi Qiu He Tian...hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of...an enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides...redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its ...

  5. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

    Type
    Blog Post
    Published
    Oct. 29, 2019, 2:52 p.m.
    ...are not always easy to track at the transcriptional or translational level. However, several genetic tools...slightly slower scale.  All living cells with a membrane possess an electrical state. This state, known...is maintained by ion channels and pumps in the membrane. An emerging interdisciplinary field of biology...features increased brightness and improved plasma membrane specificity. Stable Step Function Opsin - A stabilized...Use of genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (...PMID: 29684787. Oviedo, Néstor J., et al. "Long-range neural and gap junction protein-mediated cues control...PMCID: PMC2823934. Pai, Vaibhav P., et al. "Transmembrane voltage potential controls embryonic eye patterning...

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

    Type
    Blog Post
    Published
    Aug. 6, 2019, 12:55 p.m.
    ... integration of a novel gene randomly into their genome, some transgenic strains are difficult to breed...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous...are many types of genetically engineered mice: transgenic mice, knockin and knockout mice, and conditional...that only one chromosome needs to contain the transgene in order to see an effect. Hemizygous mice can...breed. Some problems with transgenic mice that can impact reproduction include embryonic lethality, a short...expression level, and site of integration of the transgene. These problems can be mitigated in different ...different ways. Prior to engineering a transgenic strain, the promoter being used should be tested in cell lines...

  7. Hot Plasmids February 2024

    Type
    Blog Post
    Published
    Feb. 20, 2024, 2:45 p.m.
    ...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed this problem...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy...2023). StayGold variants for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https...breaching the blood-brain barrier and delivering a transgene, with neuron bias observed in young macaques and... viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Nature...the Gradinaru lab, AAV1-X1, was developed by transferring the 7-mer peptide region from variable region...

  8. Finding Your Science Policy Path

    Type
    Blog Post
    Published
    Sept. 22, 2020, 1:15 p.m.
    ...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...variety of backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering...happen at the national level within the executive branch or in Congress, or can also happen at the state... early career researchers who have decided to transition out of academia from graduate school or a postdoc... came to DC to do either a sabbatical year or transition away from academia as tenured professors. There...taken through your own personal lens.  For me, the Veterans Health Administration was the perfect position...

  9. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....used to quantitate RNA levels using reverse transcription-qPCR (RT-qPCR). The first step of this process...requires RNA to be converted to cDNA using reverse transcription and the cDNA is subsequently quantified by qPCR...

  10. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...vivo starts with transducing cells with the CRISPR library in vitro before transplanting them into mice....time. This reduces the chance of integration at random locations that could cause deleterious effects....portions of the AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The...The good news is that this integration is not random, nor is it a global occurrence, as integration sites...target cells or tissues. AAV also shows improved transduction efficiency over lentivirus in many mouse tissues...019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022..., U., Jabaudon, D., & Platt, R. J. (2023). Transcriptional linkage analysis with in vivo AAV-Perturb-seq...

  11. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations...an additional couple of days, as it requires transduction of more cells, while AAV can be titrated following... baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called ...between insect and mammalian systems. Baculovirus transfer plasmids have a much larger packaging capacity...

  12. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...tool that responds strongly to magnetic fields (Frank Hayward et al., 2024). They deposited their plasmid...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist...when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence intensity...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license. They...mScarlet, and AsLOV2. After several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license.   ...tools for biological applications across a wide range of topics. He embraces open science, self-publishing...

  13. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    Published
    May 20, 2014, 2:06 p.m.
    ...or Förster] resonance energy transfer). FRET occurs when energy transfer from one FP (e.g. CFP) excites...classified by the emission color (emission wavelength range) FPs are usually classified by emission color as...as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP...nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - 610 nm Far-Red 625 -... ≥ 670 nm In addition to emission wavelength range, there are other traits that need to be considered... green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent Sensors: These FPs...related colors. Furthermore, most FPs  have a broad range of emission which will be detected by longer-wavelength...

  14. Why Do I Need an MTA Anyway?

    Type
    Blog Post
    Published
    May 1, 2014, 8:23 p.m.
    ...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

  15. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    Published
    Jan. 16, 2020, 2:18 p.m.
    ... dilution should fall within the range of the assay for a wide range of titers. We usually dilute our ...determine if your titers are within the expected range. We have also found that generating the standard...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...virus. It is important to note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies...per reaction. Since AAV titers tend to be in the range of 1012 to 1013 GC/mL, you must serially dilute ...million.  After making the dilutions, they are transferred to a new plate containing the mastermix which...generator, you will have to be careful about transferring your droplets from the droplet generator to ...

  16. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...PMCID: PMC6001023. Fujita, Toshitsugu, et al. "Transgenic mouse lines expressing the 3x FLAG‐dC as9 protein...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

  17. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...failed so far. What is most important is the transformation efficiency of the strain: when asking the cell...high fidelity DNA polymerase This will minimize random errors during PCR: we recommend Phusion or Q5. ...investigating the molecular biology of synaptic transmission and plasticity.     Javier García-Nafría is ...

  18. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    Published
    Dec. 2, 2014, 7:23 p.m.
    ... inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins...functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation...biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular...Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids to...considering the peer-review system of journals and grant applications, for example. However, at the same ...living for Japanese, which is not trivial, and the grant situation is currently much better than that in ...tough competition for academic positions and poor grant situation. So, young researchers need to publish...

  19. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    Published
    March 21, 2024, 1:15 p.m.
    ... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...accurate tagging. In Fluorescence Resonance Energy Transfer (FRET) applications, the dyes need to have compatible...and emission profiles to enable effective energy transfer, which is key to observing interactions and conformational... specifically to the AT-rich regions of double-stranded DNA and exhibits a blue-fluorescent light when...

  20. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    Published
    Sept. 10, 2015, 2:30 p.m.
    ...injections can be found here [5, 6]. Opsin transgenic mice Transgenic mouse lines have also been engineered...specific brain region can result in a particular neurotransmitter release or post-synaptic current. Fortunately...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector...at a similar spectrum as HR and also results in membrane hyperpolarization but unlike HR, this is achieved...[10]. Unfortunately, opsins are not actively transported and must passively diffuse so, depending on the...axonal tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience...et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing...
Showing: 281 - 300 of 801 results