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We narrowed to 328 results for: hal.1

Showing: 101 - 120 of 328 results

  1. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    ...biological questions. The two most common uses are: 1) to test the expression level in a specific system...brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which...Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a...be split into two halves, which are non-fluorescent on their own. If the two halves are in close proximity...determine the proximity of two proteins fused to the halves of the split FP. This technique is also is also...

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

    Type
    Blog Post
    ...from the paper for designing experiments with AAV: 1. Getting your viral vector into your cell of interest... administration routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest...information from a region. In the example shown in Figure 1, injected retrograde AAVs at one location result in...development, learning, and behavior poses many challenges in neuroscience with many brain regions, neuronal...

  3. Clear and Accessible Writing

    Type
    Blog Post
    ...intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words...information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient...are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification ... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a...scientific paper, you may feel it’s overwhelming or challenging. So instead, let’s focus on practicing with a...

  4. Early Career Researcher Toolbox: Social Media for Scientists

    Type
    Blog Post
    ...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ...professional development, and while it’s not challenging to use, it’s hard to know where to begin. To ...

  5. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ... to amplify away from the insertion site. Encode half the new sequence at the 5’ end of each primer, with...

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

    Type
    Blog Post
    ...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important...the power 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on ...described by the t½, which is the time after which half of the initial fluorescence intensity remains. The...

  7. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....2015; Koike-Yusa et al., 2014; Ma et al., 2015; Shalem et al., 2014; Wang et al., 2014), knock down (Gilbert... 26190106. PubMed Central PMCID: PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout...

  8. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 ...vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 ...

  9. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    ...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...stay tuned!  References Alpár, Alán, et al. "Hypothalamic CNTF volume transmission shapes cortical noradrenergic...

  10. Technical Design of a Western Blot

    Type
    Blog Post
    ...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...gel chemistry and/or specialty gel options. Table 1: Types of gels  Gel Type Protein sizes Running... Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...

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

    Type
    Blog Post
    ...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1), 21842. https://doi.org/10.1038/s41598-020-78827-...for bacterial imaging . Microbiology Spectrum, 12(1), e0369023. https://doi.org/10.1128/spectrum.03690...Biochimica et Biophysica Acta - Biomembranes, 1838(1 Pt A), 15–33. https://doi.org/10.1016/j.bbamem.2013.09.005...interchangeable labeling using the HaloTag® system, the new Janelia Fluor HaloTag Ligands expand the self-labeling... self-labeling proteins would be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme...Hayashi-Takanaka et al., 2014). Other dyes may prove challenging to stain intracellular compartments in in vivo...

  12. Adenovirus Guide

    Type
    Guide
    ...to an antigen. AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid. Adenovirus...icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over or... This construct is then transformed into AdEasier-1 cells (Addgene #16399) , which are BJ5183 E. coli ... viruses. The adenoviral backbone plasmid pAdEasy-1 (Addgene #16400) is suitable for most purposes. For...production system. Figure is based on the use of pAdEasy-1. Created with BioRender.com. AdMax™ AdMax™ is another...landscape . Signal Transduction and Targeted Therapy, 6 (1), 53. https://doi.org/10.1038/s41392-021-00487-6 (...as a Zika virus vaccine . Scientific Reports, 10 (1), 3613. https://doi.org/10.1038/s41598-020-60238-5...

  13. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    ...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology... the body or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size also provides...plasmid in bacteria (Ahmed et al., 2012). One challenge of bacterial expression is the proper formation...

  14. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; ...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors...is multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

  15. Synthetic promoter AAVs for cell-type specific expression in retinal cells

    Type
    Blog Post
    ...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...active in retinal cell types in mice. Figure 1: The four strategies used to design synthetic promoters...highest rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression ... at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different approach to ...to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...

  16. Open Resources and Plasmid Tools For Studying C. elegans

    Type
    Blog Post
    ...by splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...With the split system, only cells that have both halves of cGAL can drive expression of your gene of interest...

  17. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    ...initiating cleavage of the transcript.   Figure 1: A predicted conserved secondary structure and sequence...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic...processing and contributes to variability in RNA half-life, and ultimately gene-expression. Polyadenylation...39322032   No terminator is 100% efficient at halting transcription of the template and initiating the...

  18. An Introduction to Adenovirus

    Type
    Blog Post
    ...that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing...an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval... like other viral vectors, though significant challenges remain (Wold & Toth, 2013).  While the pre-existing...

  19. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)... of certain tumors. However, antibody stability, half-life, tumor penetration, inadequate concentration...

  20. RUBY-Red Siliques

    Type
    Blog Post
    ...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR 101... positive plants, but screening these can be challenging and requires specialized equipment. Reporters...
Showing: 101 - 120 of 328 results