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We narrowed to 199 results for: guide rna expression plasmid

Showing: 141 - 160 of 199 results

  1. Hot Plasmids February 2024

    Type
    Blog Post
    Published
    Feb. 20, 2024, 2:45 p.m.
    ...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but did...escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing of 80–90% in human cell lines...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused... al. 2023 with permission.   Find Cas-PAGE plasmids here!   Zhang, Z., et al. (2023). Efficient engineering...structured illumination microscopy in HeLa cells expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm...inspired and informed this section.  Find mStayGold plasmids at Addgene here!   Ando, R., et al. (2023). StayGold...

  2. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ...Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools... paper in Science Find the anti-CRISPR expression plasmids CRISPR-based method for cell lineage tracing...SaCas9 genome editing plasmids String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR),...between the gRNA and target. Read the paper in Development Find the Valcyrie plasmids If you have...paper in Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase...wheat. Read the paper in Genome Biology Find plasmids containing these base editors Spatially and ...

  3. Antibodies 101: Affinity Tags

    Type
    Blog Post
    Published
    Sept. 19, 2023, 1 p.m.
    ... single protein is expressed, with the intent of purification, it will be expressed along with all other...Addgene’s antibody guide Antibody protocols  Resources on the Addgene blog Plasmids 101: Protein tags...other cellular proteins within the expression system (bacteria, yeast, mammalian, etc.). To individually...about that protein that no other protein in the expression system has. This is where affinity tags come ...peptides which do not naturally exist in most expression systems that can be added to the N or C terminus...on: The protein you are trying to purify The expression system If you want the tag to remain on your ...MBP tag, although large, can actually increase expression and solubility of the tagged protein (Fox and...

  4. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    Published
    Aug. 27, 2015, 2:30 p.m.
    ...create a single gRNA-expressing plasmid, but it can also be adapted to express multiple gRNAs. Addgene has...  Check out more Plasmids 101 Posts! Check out Addgene’s restriction cloning guide! CRISPR 101: Multiplex...oligonucleotides specifying a gRNA target sequence into a Cas9-containing plasmid such as pX330. This cloning...Multiplex Expression of gRNAs Choosing a Molecular Cloning Technique  ...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method...Entry DNA overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification...fragments to create TAL effector nucleases. These plasmids were designed to utilize the BsaI and BsmBI Type...

  5. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    Published
    Jan. 16, 2024, 6:13 p.m.
    ...and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid Transduction...Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral vector containing a gRNA. These... Your gRNA for CRISPR Genome Editing CRISPR Cheat Sheat Resources from Addgene.org CRISPR Guide CRISPR...  The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer lentivirus preparations...first generation CHIME (pXPR_053). Find X-CHIME plasmids here!   Marty LaFleur is a Postdoctoral Fellow...

  6. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you... multiple parts transcribed together. They can express one or more gene(s), and typically begin with a...for more complex CRISPR experiments (e.g., many guides/parts delivered) in certain model organisms. In...Resources on Addgene.org Addgene’s Modular Cloning Guide EcoFlex MoClo Kit CIDAR MoClo Kit MoClo Plant Parts...NEB’s DAD Interview with Nicola Patron on MoClo Plasmids 101: Golden Gate Cloning ...many gRNAs delivered with a relevant Cas nuclease. MoClo can be used to assemble multiplexed gRNA systems...PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn, F., Korolev, A., Sanjurjo Loures...

  7. 3D Printing Meets CRISPR Cas9

    Type
    Blog Post
    Published
    Oct. 17, 2017, 12:55 p.m.
    ...allow the complementary base pairing between the guide RNA and the target DNA strand, and then activate two...Addgene.org Visit Our CRISPR Guide Page CRISPR/Cas9 History Browse All CRISPR Plasmids and Resources ... BioMolecular Modeling (CBM) in 1999 with the expressed purpose of creating physical models of proteins...interactive images of their proteins. Since the 1980’s, journal articles have been filled with impressive, full-color...their attention on something tangible. Under the guidance of a trained educator, these instances of focused...

  8. Fluorescent Proteins 101: Fluorescent Protein Timers

    Type
    Blog Post
    Published
    May 4, 2017, 2:30 p.m.
    ...fluorescent protein timers When FP timers are first expressed, they predominantly fluoresce one color but slowly...timers (whether as part of protein fusions or expressed independently) originally fluoresce green or blue...displays predictable kinetics over 14 hours of expression in C. elegans embryos. However, this initial ...ideal FP timers: 1. Ratiometric determination of expression time The ratio of mature to immature fluorescence...fluorescence from the FP timer is dependent on the total expression time but independent of protein concentration...Calibration curves correlating this ratio to the total expression time can therefore be used to determine how long... long a pool of FP timers has been expressed, either in whole cells or as a pool of fusion proteins localized...

  9. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    Published
    May 30, 2023, 1:15 p.m.
    ... to co-express them from one promoter. Specificity The ability of AAV to drive gene expression in specific...cell-specific gene expression and viral infection. Cre or Flp dependent switches control gene expression via switches...transgene expression, since you don’t have to wait for the host to replicate the DNA before expression. Now...non-enveloped icosahedral capsid, that can be used to express genes of interest in cell and animal models. AAVs...laboratory experiments ranging from transient gene expression in a particular cell type to CRISPR genome editing...genes, you can coinfect with multiple viruses to express genes or gene sets over 4.7 kb. Co-infection rates.... On the other hand, if you’re interested in expressing multiple small genes, you can design single rAAV...

  10. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    Published
    April 23, 2014, 1:08 p.m.
    ...four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral...lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown...majority of transfer plasmids use the weak viral LTR promoter to drive expression of the gene of interest...levels of protein expression tends to be much lower than those seen when using a plasmid backbone that was...generation systems consist of an envelope plasmid and a packaging plasmid encoding HIV proteins gag, pol, rev...generation systems require an envelope plasmid, and two packaging plasmids, one encoding gag and pol and a second...glycoprotein will be expressed on virus particles? A5: This depends on the envelope plasmid you use as the ...

  11. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via...wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects growth rate...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but...thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low (even in...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%...PMCID: PMC4262738. 3. EGFP gRNA (BRDN0000561167) (Plasmid #80034) and BRAF gRNAs (BRDN0001145345 and BRDN0001146266...

  12. Getting Started with Behavioral Neuroscience: Standardizing Experiments and Using Viral Vectors

    Type
    Blog Post
    Published
    Aug. 20, 2019, 12:31 p.m.
    ... targeting the gene is cloned into a viral shRNA expression vector and the vector particles are delivered...precisely control where and when certain genes are expressed to investigate their role in a specific behavior...Which viral vector should be used? Modifying gene expression Generally speaking, one may want to achieve either...application.  Genetic manipulation can also reduce expression levels of certain genes. A popular method uses...allows scientists to draw conclusions due to gene expression in a specific brain area, avoiding side effects... a tight spatial and temporal control of gene expression manipulation. They are injected using a stereotactic...staggered injections may be considered. Timing: High expression levels of the viral vector trangenes typically...

  13. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and...Resources on Addgene.org Browse the CRISPR guide Find CRISPR plasmids for your research ...that expressed either Cas9 or an optimized cytidine base editor. The lab then introduced an sgRNA targeting...that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-... of a mutated start codon to initiate protein expression, it can be used for an array of detectable proteins...

  14. Important Considerations When Using AAVs

    Type
    Blog Post
    Published
    June 13, 2017, 2:30 p.m.
    ... have found that expression occurs quickly and results in long and stable expression (up to 9 months in...the onset of gene expression. However, it has yet to be determined what gene expression looks like at 3 ...therapeutic molecules such as microRNAs (miRNAs), short hairpin RNAs (shRNAs), antisense oligonucleotides...from https://www.addgene.org/viral-vectors/aav/aav-guide. Where in the tissue/what cell type? In the CNS,...AAV2 can transduce many tissue types, it has low expression in several brain regions where other serotypes... under study. Many AAVs will show significant expression in a variety of cell types, but you should try...transport desirable or not desirable? If diffuse gene expression is tolerated, then axonal transport is fine and...

  15. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    Published
    Feb. 13, 2025, 2:15 p.m.
    ...background with our CRISPR Guide Browse CRISPR plasmids for base editing Find validated gRNAs ...are also RNA base editors, which you can learn more about in this blog post: CRISPR 101: RNA Editing with...activity and reduced RNA off-targeting activity compared to ABEmax. In an alternative approach, engineering...off-target activity.  Find CRISPR base editing plasmids at Addgene Improving cytosine base editing scope...editors make it into the nucleus and that they are expressed well. The Liu lab modified the nuclear localization...development of adenine base editors (discussed next!), RNA adenine deaminases have been engineered and/or evolved...team used directed evolution to create one from the RNA adenine deaminase TadA.   After seven rounds of molecular...

  16. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...Find Optogenetics Plasmids Find Biosensor Plasmids Visit the Fluorescent Protein Guide Pages...Frank Hayward et al., 2024). They deposited their plasmid with Addgene, hoping to inspire others to explore... the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable...one-component magnetoresponsive system that could be expressed and purified. Although a ~1% change in signal ...” magnetoresponse was clearly visible whether expressed in E. coli, in mammalian cells, or in purified...   Video 1: Fluorescence movie of E. coli expressing MagLOV while a magnet is waved under the sample...Lab is eager to hear from you. Find the MagLOV plasmid here!   References and resources References Duret...

  17. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ... tag plasmids here! Where should I place the tag and how many do I need? When designing a plasmid containing...Antibody Guide Ready-to-use antibodies from Addgene More Resources on the Addgene blog Plasmids 101: Protein...tag is a string of 6 or more histidine residues expressed in frame on the N- or C-terminus of a recombinant...ion substrate. If your protein of interest is expressed at a very low level, try a longer histidine tag...purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad ...isolated.   Prior to the polyhistidine tag, alternative fusion tags were used, such as S. aureus’ Protein...

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

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ... newly updated CRISPR Guide! Find CRISPR Plasmids for Your Research Browse gRNA Design Tools  ...Kaminski et al. designed gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in...portion of the LTRs joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV-1 ... cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy ...preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in higher rates of... infected with HIV-1 only after stable expression of Cas9/gRNA. Viral production is higher in this model...

  19. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    Published
    June 22, 2017, 2:30 p.m.
    ...Science Plasmid Collection Check Out Plasmids for FRET Check Out the Fluorescent Protein Guide ...A practical guide to evaluating colocalization in biological microscopy." American Journal of Physiology-Cell...into a vector encoding a fluorescent tag. Once expressed as a fluorescent protein fusion, it is possible... on the gene names in the figure below to find plasmids containing that particular gene. ... For subcellular markers in yeast see our plasmid collection from Sue Jaspersen (Stowers Institute...Prescott (Monash University). You can find more plasmids for labeling your subcellular structure of interest...interest in the Allen Institute for Cell Science Plasmid Collection. You can also use the library of full-length...

  20. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    Published
    July 11, 2023, 1:15 p.m.
    ...targeting receptor expression as a ligand and receptor pair. Targeting Receptor Expression Using chemogenetics...therapies when expression must be restricted to specific circuits. Methods for targeting expression involve ...our chemogenetics guide Find viral preps for your research Find chemogenetics plasmids for your research...target effects. For example, if your receptors are expressed in the brain, it is necessary to use ligands capable...different responses depending on what cell type it is expressed in. The most common target for chemogenetic manipulation...modify other properties such as endogenous gene expression, which can lead to changes in normal cell physiology... H, Fishman GI, Conklin BR (1999) Conditional expression and signaling of a specifically designed Gi-coupled...
Showing: 141 - 160 of 199 results