Skip to main content
Addgene
Open Menu

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

Information

In Category

We narrowed to 1,050 results for: ins;

Showing: 341 - 360 of 1050 results

  1. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    ...transposon to stably integrate a reporter gene that contains a puromycin resistance gene followed by the target...the desired genome edit:  A donor plasmid that contains a puromycin resistance gene linked to an in-frame... when transfecting your cells.  A plasmid that contains Cas9. The Madison lab also created an inducible... labor-intensive, require multiple fluorescent proteins, and aren’t that efficient. To overcome these ...named GEmCherry1, due to the “genomic fragment inserted into mCherry”. However this one mutated methionine...GEmCherry2 work? The premise is that the genomic region inserted into the N-terminus of mCherry can be targeted...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of...

  2. 3 Challenges in Plant Synthetic Biology

    Type
    Blog Post
    ...While institutions that focus on creative problem solving like the Howard Hughes Medical Institute have...technology. Monoculture and chemical spraying, mainstays of modern agriculture, are now synonymous with...control over their interaction with it, we hope to inspire and open a new conversation about GMOs.  Challenge...involves two basic components: the pigments (anthocyanins), and the plant circadian clock. Petunias have...transformation technologies like those pioneered by Ewen Mullins of Ireland’s Teagasc.  Plant biotechnology is full...business enterprises, he can be found climbing mountains or playing with his two cats. Keira Havens grew...Biophysics and Biochemistry from the Illinois Institute of Technology in 2004, she accepted a commission...

  3. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    ...digest to cut out the inserts followed by a five-piece ligation with the four inserts and the master plasmid...mammalian or other cells. TALENs are engineered proteins that comprise a DNA binding domain and an endonuclease...technology allows you to create a pair of designer proteins that will introduce a double-strand break in the...FokI endonuclease domain as well as fluorescent proteins used to sort the cells after the plasmids have...question – cardiomyocytes, adipocytes or neurons, for instance – to see how those mutations influence the disease...particular disease state. You could use the kit to insert all of those variants into stem cells with the ...introducing a mutation somewhere else in the genome. It remains to be seen which system is cleaner in terms of ...

  4. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    ...suite of plasmids (pGT) with various replicative origins that can be used for broad or narrow range of microbial...longitudinal studies of the gut microbiome, James Collins’s lab developed a paper-based method that can be...concentration within a sample. Using this system, the Collins lab was able to detect mRNA from 10 bacteria of...methods, the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit ...with broad host-range (Meyer, 2009). The vector retains the oriT for delivery into recipients by conjugation...microbial population dynamics in the gut can provide insight into how the microbiome responds to infection, ... (DCDC), relies on the assembly of fluorescent proteins into one bright particle that segregates into ...

  5. CRISPR 101: Cas9 vs. The Other Cas(s)

    Type
    Blog Post
    ...new Cas enzymes on the block? The Cas family of proteins has many useful genome engineering enzymes, each...other Cas enzymes is its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean...Unlike many other Cas enzymes, Cas14 targets ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity -...nuclease, however, unlike Cas9, it targets RNA instead of DNA. This makes it particularly useful in generating...composed of the fusion of several Cas7 and Cas11 domains. Cas7-11 cuts ssRNA and is capable of pre-crRNA...   Table 1: Fast facts on Cas family proteins and genome editing abilities   Choosing a...adaptable Cas currently available, the other Cas proteins should not be overlooked. The unique features ...

  6. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    ...constitutively expressed proteins while tet-on turns on constitutively silenced proteins. Addition of tetracycline...degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron tags, just ... to temporarily reduce expression of essential proteins (when knockouts aren’t viable). siRNA or RNAi ...gene of interest. It will inhibit transcription instead of introducing a DNA double strand break. Transient...transgene vector. Degron tags Tagging relevant proteins with a degron tag can provide inducible protein... easily implemented for exogenously introduced proteins, as many of them just need a special promoter ...

  7. RNA Extraction Without A Kit

    Type
    Blog Post
    ...enzymes that degrade RNA, are especially hardy proteins that are found in and on everything, including...solution and spun down the homogenate to remove the insoluble material. Then, the homogenate was loaded onto... hours to separate the intact RNA from DNA and proteins. Although very effective at isolating total RNA...RNA could be effectively separated from DNA and proteins by a simple extraction protocol with guanidinium...in a guanidinium thiocyanate solution. However, instead of RNA separation using cesium-chloride gradients...in the top, aqueous layer, while DNA and other proteins are retained in the interphase and bottom, organic...into one step. After homogenization in TRIzol®, insoluble material is removed via centrifugation and the...

  8. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    ...researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach is not...Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed targeting capacity. By using ...two hairpin sequences, which recruit fluorescent proteins BFP, GFP, and RFP fused to the respective hairpin...and red. In contrast, a gRNA with two different hairpins will produce a secondary color cyan, yellow, or... individual colors to 6. A gRNA with all three hairpins generates white light. All 6 gRNAs can be expressed...composite. gRNAs with two identical hairpin binding domains produce blue, green, or red fluorescence, depending...Learn Some of the Pitfalls When Using Fluorescent Proteins Control Protein Activity with GFP Resources on...

  9. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ...However, instead of weakening interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted..., researchers from the Zhang lab at the Broad Institute, the Joung lab at Harvard Medical School, and ...groove formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and ...specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly thought that...mutations N497A, R661A, Q695A, and Q926A (SpCas9-HF1 contains all four mutations). Although the precise mutations...from Addgene here! Future possibilities for Cas proteins With their enhanced specificity, eSpCas9, SpCas9...25079318. PubMed Central PMCID: PMC4176945. 4. Kleinstiver, Benjamin P., et al. "High-fidelity CRISPR–Cas9...

  10. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

    Type
    Blog Post
    ...Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of human proteins that...past, we've been working with the Rare Genomics Institute to provide plasmid grants to researchers working... his lab’s research is the mechanisms by which proteins assemble into functional multi-subunit macromolecular...function of even a small fraction of Arg-mutant proteins could improve health outcomes for millions of ...treatment for rare diseases caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant...large number of other disease-linked Arg-mutant proteins both in yeast and human cells in order to determine...rescue of a variety of disease-linked Arg-mutant proteins in the lab, clinicians might consider prescribing...

  11. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...icosahedral capsid made of 3 types of proteins: fiber, penton and hexon proteins. The viral genome DNA is further... which contains the adenoviral genes necessary for virus production. For detailed instructions on how ...consisting of a capsid surrounding an inner core which contains the adenoviral genome. The genome itself is a ...further protected by histone-like proteins forming the nucleocapsid. B) Three generations of adenoviral ...ceases relatively quickly due to immune response against AdV. Importantly, recombination-competent adenovirus...helper adenovirus is required to provide the viral proteins in trans. The Gutless AdV have generated high ...

  12. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    ...at the Korea Advanced Institute of Science and Technology (KAIST) and Institute for Basic Science (IBS...protein. After fusing the CRY2 and CIB1 domains to separate proteins of interest, a researcher can cause ...robust and efficient oligomerization of target proteins in response to blue light. This tool, CRY2clust...2017. PubMed PMID: 28288122. New fluorescent proteins for use as FLIM-FRET acceptors  Listen to the...(YFP) with an excitation peak of 519 nm. These proteins have improved photophysical properties and can...maturation kinetics than other dark fluorescent proteins, it has good signal stability allowing for minimized...

  13. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    ...precise insertion of a DNA sequence ranging from a single nucleotide change to large insertions like the...MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the authors, they named...polyprotein Nanoblades are formed when viral structural proteins, such as the MLV protein Gag, multimerize and ...cleaves the proteolytic site connecting the two proteins, thus releasing Cas9. Nanoblades can also deliver...loaded with 1-4 different gRNAs. Viral envelope proteins To alter the cell tropism of Nanoblades, different...alternative method for generating transgenic mice strains and for non-invasive in vivo gene editing. The ...viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins." Nature communications 10.1 (2019): 45. ...

  14. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

    Type
    Blog Post
    ...detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield, 2014). Traditionally,...Therefore, researchers from over 100 scientific institutions have proposed a shift to recombinant DNA-based...expression. If starting from a protein sample (for instance, affinity purified antibodies from a patient’s...the line and subsequent cloning of the antibody chains (Crosnier et al., 2010; Andrews et al., 2019). ...well in animals because of similarities to host proteins. After the antibody of interest has been cloned...non-antibody based affinity reagents (e.g., monobodies, DARPins) (Helma et al., 2015).    The Many Benefits of...and sequences for antibodies are not shared. For instance, through open plasmid sharing, the CRISPR community...

  15. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...shipping materials (i.e., a deposit kit) with instructions on how to send liquid DNA or bacterial streaks...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose...plasmid will work for them. Species of Gene or Insert Choose from: H. sapiens (human), M. musculus ...luciferase, cre/lox, yeast expression, worm expression, insect expression, plant expression, mouse targeting, ... cannot be propagated in any of these standard strains, choose other, indicate strain in the “Growth Strain...showing the first eight columns and a pop-up instruction box for the “Purpose” field. If you ever have...should go into a cell, hover over the cell and instructions will pop up. When you’re finished filling out...

  16. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    ...student and then instructor at Singularity University at NASA Ames, and an instructor at Biocurious.org...makerspaces around the world. Hackerspaces.org maintains a list of active spaces near you.   Learning ...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...Minnesota has declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00...motor behaviour, and ultimately submit our new strains back to the CGC for other researchers to use. Although...equipment that would otherwise be discarded. For instance, the centrifuge in the picture had a broken controller... the fantastic optics and fluidics platform it contains. Biotech equipment becomes obsolete amazingly ...

  17. Streaking for Single Colonies: The Streak Plate Challenge

    Type
    Blog Post
    ...add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked the students...perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent protein family...off color in ambient light. Expressing these chromoproteins in E. coli would make it easy to distinguish...single colonies, these chromoprotein-expressing strains are easily amenable to other experiments to teach...have the students transform them into different strains of E. coli,” says Westenberg. During the transformation... how different genetic backgrounds of the host strains affect transformation efficiency.  Aside from the...blog Read our other blog posts on fluorescent proteins Check out educational resources on the blog Resources...

  18. Plasmids 101: Degron Tags

    Type
    Blog Post
    ...modulation of endogenously and exogenously expressed proteins. Here we will review what advantages degron tags... the tagged protein. Degrons are destabilizing domains which will be identified by various enzymes and...manipulate endogenously and exogenously expressed proteins. Thirdly, degrons and their drugs are titratable...not amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag self-...Promega as an affinity tag for visualization of proteins of interest. A fluorescent Halo ligand can be ... molecules which target E3 ligases to specific proteins by simultaneously binding both their target protein...their effectiveness with over a dozen different proteins. In the end, you can’t predict with absolute certainty...

  19. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

    Type
    Blog Post
    ...coding genes in the human genome with fluorescent proteins. For each gene, we needed to design guide RNAs...ranked by specificity of targeting or by cut-to-insert distance for HDR. (The notable exception is TagIn...compressed CFD score) * (gaussian normalized cut-to-insert distance) This gives each guide a score ranging... can be used in the rank equation above. Cut-to-insert distance was fitted to experimental data of its...the greater this distance between the cut and the insert, the more poorly the guide performs HDR. Optimal...sequences to prevent re-cutting of the sequence inserted by HDR. Although this practice is generally advised...enough to prevent re-binding. Lastly, the sequence inserted by HDR may inadvertently re-create a match for...

  20. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ... pC5Kan-P2A P2A Insect pUltra P2A and T2A Lentiviral Ac5-STABLE2-neo T2A Insect   For the co-expression...vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters ... detect your gene of interest, scientists have instead developed novel methods to co-express your gene...IRES Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation...site, thereby resulting in co-expression of two proteins from a single mRNA. IRES was originally discovered...between your genes of interest and you can then insert the whole multicistronic cassette into a backbone...Although it is recommended to use the 2A peptides instead of an IRES when stoichiometrically equivalent levels...
Showing: 341 - 360 of 1050 results