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We narrowed to 833 results for: ins

Showing: 161 - 180 of 833 results

  1. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ...cells. The Brainbow-1.0 construct contains three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP...injection to insert these Brainbow constructs into mice. When they imaged the mouse brains, they found ...Brainbow-1.1 scales up the system to four fluorescent proteins, with recombination events specified by three ...colors (n-GFP, RFP, YFP or M-CFP.) The construct contains two tandem invertible segments, each with two ...The remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system,...intensity, caused partially by fluorophore photoinstability, as well as the tendency of fluorophores to... First, they screened a variety of fluorescent proteins to find those with ideal characteristics (low ...

  2. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    Published
    April 6, 2021, 12:30 p.m.
    ...the gene/insert by aligning to NCBI entry or using BLAST.   Confirming tags and fusion proteins. We’ll...of the plasmid.   Confirming the insert We usually confirm the insert through BLAST or by direct alignment...Confirming tags and fusion proteins Finally, we confirm promoters, tags, fusion proteins, and selectable markers...and user friendly, to own the process entirely remains a large undertaking. Priority number one was to...by the depositor. Depositing labs often provide insert sequences or annotated Genbank files that are useful...We look for point mutations, truncations, and insertions that could compromise function. When we do find...promoter, and rabbit beta-globin splice acceptor, contains sequence that is over 80% GC. Some IRES sequences...

  3. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    .... Inhibitory opsins are usually light-driven pumps that pass ions (Cl-, H+, Na+) against the concentration...Light-gated ion channels Light activated ion channels (rhodopsins) either excite or inhibit neuronal activity upon... of light (Vierock et al., 2021). Step-function opsins are chimeric ion channels that turn on with a light-pulse...are many different opsin (natural and engineered proteins responsive to light in combination with retinal...activation inhibitory (ArCH) proton pump passing ions against the concentration gradient. Created with BioRender.com...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate...partners such as light, oxygen, and voltage (LOV) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2...

  4. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    Published
    Feb. 8, 2018, 12:17 p.m.
    ...fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins to make ... imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs) can be problematic...fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging tools but their use is limited...tissues. To avoid absorbance of heme-containing proteins in whole animals, light at or above 600 nm wavelengths...fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited by Nluc emission and the lab has...maturation time compared to similar orange fluorescent proteins. In their work, the authors noted how CyOFP1 could...of the Enhanced Nano-Lanterns to other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase...

  5. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    Published
    Oct. 11, 2018, 12:30 p.m.
    ... the overhangs of the assembled inserts. After ligation of the insert into the vector, the BsaI site is...transformants by colony PCR confirmed the insertion of the Lambda insert at the same high frequencies as the...single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway... BsaI, and a DNA ligase such as T4 DNA ligase. Inserts, either precloned or in amplicon form (shown above...ordered, pre-determined manner. If the original insert religates into its original vector, it will subsequently... assembly: High efficiency and accurate single-insert assembly Intermediate 5- or 12-fragment assembly...assembly More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...

  6. Anatomy of a Plasmid Page at Addgene

    Type
    Blog Post
    Published
    Feb. 4, 2016, 3:30 p.m.
    ...depositing lab page contains all the plasmids from the same lab while the publication page contains all the plasmids...s plasmid number here. Gene/insert: This describes what has been inserted into the backbone, and provides...the gene/insert, this information can be found here. There may be more than one gene inserted into a backbone...associated, such as when a plasmid contains certain fluorescent proteins. Finally, for many plasmids you ... backbone, and each is given its own gene/insert section on the plasmid page. Tags are listed according...or C terminus of the transcribed and translated insert. If it is an empty vector with a tag, it’s terminus.... coli lab strain DH5a, but others may require strains such as ccdB survival (for Gateway plasmids).  ...

  7. Zebrafish as a Model for Behavior: Swimming into the Optogenetic Spotlight

    Type
    Blog Post
    Published
    July 16, 2019, 12:59 p.m.
    ...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers...optogenetics.  Common opsins used in zebrafish Optogenetics functions through opsins which are light sensitive...sensitive proteins. Opsins used in optogenetic studies are not native to vertebrates and are commonly introduced...fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks of life...system for behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...

  8. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    Published
    Nov. 1, 2016, 2:30 p.m.
    ... primers used to amplify the insert have sequences specific to the insert with an additional 15-17 bases...and insert. Once you’ve confirmed that the PCR was successful, mix together the vector and insert PCR ...Connecticut. Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without... of specific sequences for base pair overhangs, insert and vector purification, and, most importantly,...independent, it does not require purification of insert or vector products, nor does it require the use...In FastCloning, you PCR both the vector and the insert in separate reactions and then combine the products...procedure To start the FastCloning process, the insert and vector are first PCR amplified. The forward...

  9. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...well — namely that they can cause cancer via insertional mutagenesis (which HIV does not do). Furthermore... yet retroviruses are RNA-containing viruses  (Robinson et al., 1965)! You may be beginning to realize...the fact that the virus must have its structural proteins expressed from a reverse-transcribed piece of ...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... The RT protein is made by a separate set of instructions in the same producer cells as the virus. Because... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...

  10. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...optimizing strains with low efficiency.       Sheng Yang obtained his Ph.D. from Shanghai Institute of Biochemistry...2015). In addition to the pMB1 replicon, pTargetF contains the sgRNA targeting the genomic site of interest... system.   Figure 1: The editing process begins with the addition of the sgRNA sequence in pEcgRNA...method was much simpler and took only ~32 hours instead of ~60 hours. We successfully tested the pEcCas...precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade and pCutamp...

  11. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    Published
    April 14, 2016, 2:30 p.m.
    ...distances. For instance, how much force can be generated by conformational changes in proteins? How can information...produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance...rupture E. coli spheroplasts to release encapsulated proteins. Video by ACS. For a more cartoony explanation...rearranging into different polymerization states. Instead, R body monomers respond to pH changes by undergoing... variety of conditions. They are composed of 4 proteins encoded in a single operon and were first expressed...

  12. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

    Type
    Blog Post
    Published
    March 10, 2015, 2:25 p.m.
    ...Antibodies are composed of two light chains and two heavy chains. The ends of the branches contain the...distribute monoclonal antibodies against popular fluorescent proteins (GFP and YFP) and protein tags (...binding specificity allows scientists to target proteins of interest for labeling, quantification, purification...cell lines. The DSHB was created by the National Institute for Child Health and Human Development (NICHD)...distribution funds our operation. Antibodies are glycoproteins that are part of the humoral immune system. ...hybridoma lines, each producing a monoclonal antibody against a target antigen. This contrasts with polyclonal...popular monoclonal antibodies include those directed against human transcription factors such as PAX7, PAX6,...

  13. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...pull-down assays: Epitope tagging provides a solution Proteins - the workhorses of the cell – never work alone.... It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform...complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes... mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments...for each target protein. To study 20,000 human proteins, this would be time-consuming, expensive, and ...biochemical “epitope tag” such that these tagged proteins can be immunoprecipitated using a standard tag-specific...assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed...

  14. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming... benefit of working with surface proteins is also that such proteins are easily studied in living cells...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest...screen, each gene is not targeted by only one gRNA, instead many different gRNAs are used for each gene. At...molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different contexts.... Thus, a screen can be used to target surface proteins to determine which contact points are involved... used to interfere with the identified surface proteins, and such antibodies could thus be interesting...

  15. Plasmids 101: Biotinylation

    Type
    Blog Post
    Published
    Nov. 15, 2018, 1:50 p.m.
    ...effectively biotinylate AviTag expressing proteins in bacteria, yeast, insect and mammalian cells. Check out Kay...about in vivo biotinylation of bacterial fusion proteins Biotin is an essential vitamin for life Although... immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns...help with the specific elution of biotinylated proteins. For help determining which biotinylation reagent...Biotinylation Reagent Selection Tool. AviTag and BirA Proteins can also be biotinylated by exploiting the specificity...Kay et al., 2009 for methods on biotinylating proteins with BirA. You can find plasmids that contain ...Scientists have commonly used this method to attach proteins or polypeptides to a solid substrate (Holden and...

  16. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    Published
    April 4, 2024, 1:15 p.m.
    ...cells, live cells, my marker stains of interest). Let’s double-click inside our gate and proceed to the...present that vary in cell type, size, and marker proteins. But even if you are analyzing a homogenous cell... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage... the magnitude of FSC, SSC, and all fluorescent stains. The cytometer's software will allow you to adjust...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...where multiple cells were clumped together. (B) Instead of FSC, one is free to use SSC for a single cell...separation pattern of positive vs. negative signal remains the same (Figure 5).   A.                                                                                                        ...

  17. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ... 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific...anhydrotetracycline (aTet), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting... point mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is.../pEcgRNA, allows it to be used in more E. coli strains including the popular BL21(DE3) (Li et al., 2021...tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of... both systems, custom spacers/gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid...plasmid can be also linearized with XbaI to insert extra elements, like a repair template, using Gibson...

  18. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    Published
    Sept. 6, 2016, 2:30 p.m.
    ...refers to 3 proteins (H-Ras, K-Ras, and N-Ras) encoded by a family of proto-oncogenes. Ras proteins are special...” shape and lack of “druggable” domains. In other words, Ras proteins naturally lack areas that are accessible...Resources National Cancer Institute: Cancer Statistics Page National Cancer Institute: Ras Pathway v2.0 Page...products. Proto-oncogenes consist of a group of genes/proteins that, when expressed, normally encourage cell ... right places. Checks and balances: protecting against cancerous cells Luckily, there are several safety...not ideal environments for many cell types. From proteins to pathways - Ras as a model When mutations do...different cancers and identifying the roles of key proteins involved, with the goal of being able to discriminately...

  19. Build Your CRISPR Vocabulary

    Type
    Blog Post
    Published
    July 2, 2024, 1:15 p.m.
    ...most famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well, both natural...you. We’ll cover the common terminology for the proteins, DNA, and RNA used in CRISPR.   Figure...tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed...spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs...Cas: Different CRISPR systems involve different proteins, but many of them are called Cas, short for “CRISPR-associated...natural and engineered. What these proteins all have in common is the ability to use an RNA guide to target...Target strand: The strand of foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand...

  20. Course-Based Undergraduate Research Experiences (CUREs)

    Type
    Blog Post
    Published
    Feb. 9, 2023, 2:15 p.m.
    ... 13(4):602-606. Brian, D. 1996, Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  ...their trainees’ very way of thinking too. Albert Einstein might have put it best when he stated, “the value...metabolic pathways (precisely the type of exercise Einstein might have resented!). In 1997, Joseph Provost...structure, function, and interaction with other proteins.   It is important to note that research ...Kimball et al., 2020). Benefits and Challenges CUREs inspire students to take ownership of their work and become... him a more effective motivator (“be like Tony Robbins!” he encourages faculty mentors.)  Collaboration..., Provost partnered with colleagues at other institutions to form a support network called the MDH CUREs...
Showing: 161 - 180 of 833 results