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We narrowed to 834 results for: ins;

Showing: 221 - 240 of 834 results

  1. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...product to insert, and tools for primer design, so it’s always best to check in with the instructions from ... Gibson and colleagues at the J. Craig Venter Institute in 2009. Here at Addgene we added this option ...fragments, but can also be used in basic cloning of an insert into your vector of choice as shown in the figure...incubated together with an enzyme master mix, which contains three different enzymes: an exonuclease, which...using Gibson assembly, imagine that you want to insert your gene of interest into a vector with a large...included in the vector you want to use. If you inserted these two pieces of DNA by restriction enzyme ...fragments. However, by using Gibson assembly, you can insert both the gene of interest and the tag sequences...

  2. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...allowing a spacer-repeat “brick” to be inserted. The BsaI site remains intact, allowing subsequent “bricks...make sure that each cell that gets the plasmid contains all of the desired gRNAs. This increases the chance...the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two U6 promoters. To use ...available from the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods....)-containing plasmids. Each destination vector contains GFP, enabling you to select cells with high GFP...enables users to express up to 7 gRNAs. The kit contains different destination vectors depending on the...through Golden Gate or Gibson Assembly. gRNAs are inserted into the pCBC vectors using BsaI, and promoter-gRNA...

  3. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    Published
    May 19, 2020, 1:15 p.m.
    ...include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane...membrane proteins. Although all viral components are available in bacterial expression plasmids, glycoproteins...glycoproteins and other proteins that require post-translational modification may not be easily expressed ...level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility... doing things. These new processes have been instrumental in achieving our mission to help science move...Virus Entry Mediated by SARS Coronavirus Spike Proteins. In: Methods in Molecular Biology. Humana Press...

  4. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ... 100 kD proteins or 20, 40, 60, and 80 kD proteins. Plasmids containing 150 or 250 kD proteins are also...expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins run as ladders...the Genetic Pertubation Platform at the Broad Institute have expanded the genetic screening toolkit by...express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli, either individually (~20,000 lanes...also available separately. The proteins, which are purified via histidine tags, are optimized for SDS-PAGE...Western blots via engineered immunoglobulin binding domains.  Santilli, R.T., et al. Sci Rep (2021). https... large size of CRISPR-Cas effectors and fusion proteins, such as Cas9 and Cas12a, creates challenges for...

  5. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

    Type
    Blog Post
    Published
    Jan. 31, 2019, 1:32 p.m.
    ...mice, exosomes expressed specific integrins (cell-adhesion proteins) targeting different organs. This ...action outside of tumor cells, and recent studies (Martins et al., 2018 and Xing et al., 2010) revealed precisely...physiology is deeply connected to these 3 stages and contains multiple cell types: fibroblasts, myofibroblasts...whole, microenvironments promote tumor durability instead of repressing them, making it even harder to cure...possibly inducing the secretion of growth promoting proteins, such as human growth factor, vascular endothelial...microenvironment itself. They deliver compounds like proteins and nucleic acids from cancer cells to the microenvironment...microenvironments! Take myoepithelial cells for instance. They retard tumor formation by inhibiting cell...

  6. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    Published
    July 14, 2021, 1:15 p.m.
    ...recognizing the target protein remains bound to the column while the rest of the proteins are eluted. Then, the...full-length protein that you want to generate antibodies against, or a unique peptide sequence derived from this...the serum are separated, and the serum - which contains the antibodies - is further processed to isolate...Protein G purification is one common method. These proteins bind the IgG heavy chain. Running the serum over...specific to the immunogen or not, while any other proteins pass through. However, protein A and protein G...Antibodies that don’t cross-react with the serum proteins pass through the column, resulting in a polyclonal...or select a monoclonal or recombinant antibody instead. Otherwise, you might experience variability in...

  7. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...amount of information regarding which regulatory proteins, signaling molecules, mutations, and environmental...represented in a very small number of registers. Instead, one may use qPCR to determine the fraction of ...combination of red, blue, and green fluorescent proteins in each state. The three methods of state interrogation...tell you the distribution of states in a sample; instead, it will suggest the predominant state of registers...plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream of inducible...copy, bacterial artificial chromosome backbone) contains the register. Depending on the desired application...endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to...

  8. Building Global Connections with the International Mentorship Program USA-EUROPE

    Type
    Blog Post
    Published
    Nov. 6, 2015, 3:30 p.m.
    ...Massachusetts Institute of Technology, Boston University, Johns Hopkins University, the Salk Institute, and the... the Karolinska Institutet, among others). Mentors and mentees held scheduled monthly progress video conferences... confined by birth country or financial means. Instead, talent should be realized through equal opportunities...internationalization faces several obstacles; for instance there are few resources to help get individuals...supported, and can more fully identify with and be inspired by the mentor’s successful example. For evidence...2014-15, University of Valencia): “It is very inspiring to be in contact with mentors who are at a professional...universities and high level public and private institutions also showed their support and hope for the IMP...

  9. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    Published
    Nov. 14, 2024, 2:15 p.m.
    ...long ncRNAs is impractical. Instead, tags or stop elements can be inserted to disrupt the transcription...RNAs (ncRNAs) are transcribed but do not encode proteins. They play important roles in many cellular and...permanently disable, protein-coding genes by generating insertions or deletions in the DNA sequence. This same approach...break works well because very small deletions and insertions in the DNA sequence can completely disrupt expression... an open reading frame, so small deletions or insertions may not result in a functional loss (Ho et al...the ncRNA (Hazan and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription...exon or a large genomic fragment. (D) Knock-in: insertion of a synthetic polyadenylation (spA) signal. Figure...

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

    Type
    Blog Post
    Published
    July 18, 2019, 12:55 p.m.
    ...sharing their worm strains through the Caenorhabditis Genetics Center (CGC) which maintains and distributes...each half to a gp41-1-N-intein. Split inteins are protein domains that naturally “associate, self-excise...distributes the strains all over the world. When I was at the 22nd International C. elegans meeting, I was again... the Arizona State University (ASU) Biodesign institute developed a 3’UTR database specifically for C.... discovered in plants to conditionally deplete proteins of interest in C. elegans (Zhang et al., 2015)...modified TIR-1 protein can degrade degron-tagged proteins in an auxin dependent manner. This system allows...scientists to both spatially and temporally knock-down proteins of interest.  Plasmids for cGAL and split cGAL...

  11. Hot Plasmids February 2024

    Type
    Blog Post
    Published
    Feb. 20, 2024, 2:45 p.m.
    ...AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery Recombinant anti-FLAG...COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under CC-BY...for your next experiments, they will also likely inspire further-improved versions. Thanks to Christophe...Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this section.  Find mStayGold plasmids...2023). Bright and stable monomeric fluorescent proteins derived from StayGold. ResearchSquare (Preprint...alchemy: CAP-Mac unleashes the secrets of primate brains with brilliance By Hridaya Patel (Addgene co-op...systemic gene delivery in non-human primate (NHP) brains.  Systemic gene delivery capsids previously identified...

  12. Our Most Popular Blog Posts from 2019

    Type
    Blog Post
    Published
    Dec. 31, 2019, 2:26 p.m.
    ...PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection by Joachim...optogenetics, chemogenetics, plasmids, fluorescent proteins, careers, and more. These resources can now be...Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley, Shreya Vedantam, Michelle Cronin...used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi by Jennifer Tsang...and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for ...Fluorescent Vegetables in Aptamer Soup by Aliyah Weinstein RNA aptamers, which are nucleic acid sequences...shares some strategies for generating GFP fusion proteins. From considering protein size and shape, linker...

  13. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    Published
    Oct. 6, 2015, 2:30 p.m.
    ...plasmid's journey begins First, each package is opened and the contents are carefully inspected to verify that...different strains of E. coli that can be used for transformation. The preferred E. coli strains for storage... then the journey of transformation and storage begins. Some samples are submitted as bacterial colonies...the year (see graph below). Addgene scientists inspect each sample and make sure they have been barcoded...assigned to a location in a metal freezer rack that contains five levels that can hold five plates on each ... back into because every barcode on every tube contains information about that tube’s appropriate location...been received and stored, it is put into E. coli strains that have been treated to easily take in plasmids...

  14. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    ...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...amino acids found in all proteins, so you can't distinguish the different proteins from each other. To distinguish...Lastly, there are fixable dye stains that react with free amines on and inside the cells and are thus typically...distinguish different target proteins in your flow analysis, you'll need to use fluorophores that attach...appropriate readout. A common method of labeling proteins is to fuse them with a fluorescent reporter, such... reporter's fluorescence. Another way to label proteins is through treating cells with fluorophore-conjugated...antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow cytometry experiment are known...

  15. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    Published
    Sept. 10, 2015, 2:30 p.m.
    ... of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural Research Program (NIH-NIDA...are numerous variants for the different types of opsins available and their differences and uses have been...discussed thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University...details about types of constructs available, instructions for obtaining a material transfer agreement,...published at this point.  In brief, some standard opsins (see table above) include Channelrhodopsin2 (Chr2...and subsequent firing of an action potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride...specific opsin expression. However, the types of opsins you are able to use are obviously limited by the...

  16. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ... Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 is an enormously...and former researcher at the Wyss Institute for Biologically Inspired Engineering.     Alejandro (Alex)...prevent your gene from being up-regulated. For instance, generally, the more highly expressed a gene is...similar tools were made using other DNA binding proteins such as zinc fingers (ZF) and TAL effectors (TALE...scale CRISPR-based screens can be used to gain insight into protein structure and function (Pan et al....Ansari R, Harper S, Jackson DA, McRae R, Pooley R, Wilkinson P, van der Meer D, Dow D, Buser-Doepner C, Bertotti...Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated...

  17. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    Published
    April 25, 2023, 1:15 p.m.
    ...and find an ideal antibody against Protein A and an ideal antibody against Protein B. The antibodies are...isotype? Antibodies are composed of light chains and heavy chains made up of variable regions and constant...interact. You read extensive research on the two proteins and come up with a great experimental plan that...detect Protein B and successfully visualize both proteins.     Figure 2: Two primary antibodies ...In most cases, the resulting chimeric antibody retains the binding specificity to the original target ...target and can be used with secondary antibodies against the new Fc region. In addition to increasing multiplexing...     Morelock, M. M., Rothlein, R., Bright, S. M., Robinson, M. K., Graham, E. T., Sabo, J. P., Owens, R.,...

  18. Identifying Sequence Elements with SnapGene's Feature Database

    Type
    Blog Post
    Published
    Dec. 21, 2017, 2:06 p.m.
    ...microbial strains. It proved to be impractical to catalog every variant of a feature. Instead, we identified... days or even weeks. Primer design was done painstakingly by hand. Records of plasmid construction were...their goals as intuitive and painless as possible. Instead of crowding the interface with every possible option... antibiotic resistance markers and replication origins, but there is extensive heterogeneity in the feature...efforts, such an algorithm has limitations. For instance, it could miss a common feature if the sequence... inaccurately. The best example is fluorescent proteins, which often come in closely related versions ... genomes of newly sequenced bacterial or viral strains. We are working on a way for SnapGene users to ...

  19. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    Published
    Jan. 26, 2023, 2:15 p.m.
    ... generally preferred for large insertions such as fluorescent proteins or selection cassettes. This class...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Quadros RM, et...Stroik January 24, 2023. DNA damage drives genome instability and contributes to cancer, premature aging, and...sticky or blunt ends and often introduces small insertions and deletions along the way. HDR uses homologous...overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for ...template to create mutations HDR donors can contain insertable sequences or specific mutations, along with homologous...target sequence to be modified. In general, the insertion sites of the modification should be very close...

  20. March for Science

    Type
    Blog Post
    Published
    April 21, 2017, 2:30 p.m.
    ...apolitical process for seeking knowledge. The process begins with a testable hypothesis - an educated guess ...scientists then revise their hypotheses and the cycle begins again. No part of this process is inherently political...States government? Politics and science are intrinsically linked: governments exist in reality and science...Environmental Protection Agency, and the National Institutes of Health endanger scientists’ ability to uphold...consequences While frustration and anxiety have inspired people to rally for science, others are concerned... themselves can come to conclusions themselves instead of depending on scientists or politicians to interpret...lines (18). Feeling like an apple will fall up instead of down when dropped will not make it happen. In...
Showing: 221 - 240 of 834 results