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We narrowed to 916 results for: tro

Showing: 261 - 280 of 916 results

  1. A Needle in a Base-Stack: Cas9 Structural Biology

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    Blog Post
    ...very weakly, most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, ... present in the sample. Other techniques, like electron microscopy and FRET, can offer more information...well described in the closed conformation, recent electron microscopy studies have also found Cas9:RNA complexes...loops 2 and 3 are not strictly necessary for in vitro cleavage, but they improve cutting efficiency and...pairing in the seed sequence.   Figure 6: Electron microscopy structure of S. pyogenes Cas9 bound...structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy structure (right, PDB ID 7S4X), viewed...conformational state of the HNH domain also allosterically controls RuvC activity through a hinge region between the...

  2. Save Time with Transient Plant Leaf Transformations

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    Blog Post
    ...tobacco. Studying gene function in C. roseus is strongly limited by the availability of methods. Infection...check for GUS-like activity of your tissue. Run a control with Agrobacterium lacking the GUS gene since plants...promoter activity, for example. Useful positive controls could include: pSB96 - pL2_pSB90_2x35S::fLUC-I...days in the dark after the transformation. Use introns Plant promoters can be recognized or leaky in Agrobacterium...Agrobacterium, but not the plant. For example, we have seen strong expression of the GUS gene with the commonly used...Agrobacterium. This can be easily avoided by the use of introns, which will be removed from the transcripts in ...plants, check out this interview with the Nicola Patron, who shared the MoClo plant kit via Addgene. For...

  3. New Tools Enable CRISPRa for Neuroscience Applications

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    Blog Post
    ...spatiotemporal control over its expression. Constitutive or inducible Cre recombinases can be introduced by AAV...SPH mice CRISPRa has largely been limited to in vitro settings due to the large size of Cas9 activators...in vivo, they should be screened for activity in vitro and then packaged into AAV for in vivo delivery....developed neuron-optimized lentiviral vectors for in vitro and in vivo CRISPRa (Savell et al., 2019). This ...immediate-early gene Bdnf, which encodes brain-derived neurotrophic factor (BDNF). Bdnf is expressed early in response...of other CNS cell types, including microglia, astrocytes, and oligodendrocytes, provided that reporter...preferable for individual gene activation in select in vitro and in vivo applications, the tools described above...

  4. Plasmids 101: Shuttle Vectors

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    Blog Post
    ...the vector to survive while the auxotrophic cells die. Some auxotrophic selection markers can be used across...autonomously replicating sequence (ARS), a partial centromere sequence (CEN), both, or neither, depending on...autonomously replicating sequence (ARS) a partial centromere sequence (CEN) both or neither ...depending ...choose to ensure it works in your host species! Auxotrophic selection Another option is to use a different...different method of selection entirely. Auxotrophic selection relies on the host organism being unable to synthesize... example, in both S. cerevisiae and E. coli. Auxotrophic selection is especially common in yeast experiments...spontaneously develop resistance to selection toxins. Auxotrophic selection is useful for producing stable mammalian...

  5. Important Considerations in Optogenetics Behavioral Experiments

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    Blog Post
    ...  Historically, ICSS was done by implanting an electrode and non-specifically electrically stimulating ...opposite chamber) [2]. Laser activation is usually controlled automatically using motion capture software such...apply. Testing stimulation First, In lieu of electrophysiology, it would be convenient to know if you are...targeted brain region after stimulation compared to controls without Chr2. c-Fos is a reliable and widely used...easiest way to resolve these issues is with electrophysiology. Concluding thoughts and moving beyond the...PMC4181379. 3. Kheirbek, Mazen A., et al. "Differential control of learning and anxiety along the dorsoventral ...silencer: development and application to optogenetic control of non-human primate cortex." Frontiers in systems...

  6. Save Time and Money by Making Your Own Competent Cells

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    Blog Post
    ...chemically competent cells (protocol)  Preparing electrocompetent cells (protocol)  Untergasser Lab Preparation... competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene, we use the...Transforming chemically competent cells (protocol)  Electroporation (protocol)  Untergasser Lab Transformation...competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared using ...with ampicillin resistance.  After a plasmid is introduced to bacteria, the bacteria is typically grown ...competent cells, don’t forget to do a negative control (plate your cells without adding plasmid) to make...have contamination.  Also conduct a positive control (test a plasmid that transforms well in other competent...

  7. Let There Be LITE Plasmids

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    Blog Post
    ...Under those optogenetic controls, gene expression levels reliably increased in vitro by a factor of 10 to...what the LITEs now enable. In a Nature paper introducing the optogenetic tool, Konermann and Zhang showed...populations. They demonstrated use of the LITEs to control gene expression in mouse neurons and in the brains...both quickly and significantly. Genes under LITE control showed a rise in transcription in as little as ...to establish it as “a novel mode of optogenetic control of endogenous cellular processes." The LITE system... Reference: Silvana Konermann et al. "Optical control of mammalian endogenous transcription and epigenetic...

  8. Plasmids 101: Biotinylation

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    Blog Post
    ...extremely strong affinity for one another. Actually the bond between them is one of the strongest noncovalent...that scientists began to exploit biotin and its strong interaction with avidin for molecular biology and...papers and webpages are listed below: EMSA (Electrophoretic Mobility Shift Assays) ELISA (Enzyme-Linked... biotinylate macromolecules of interest both in vitro and in vivo. Biotinylation reagents Biotin’s exposed...attached to a photoreactive arylazide group. Under strong visible light, the arylazide group becomes reactive... Krijgsveld, J., Katsantoni, E., Heck, A., … Strouboulis, J. “Efficient biotinylation and single-step ...Schwartz, S. A. “Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA...

  9. Identifying Sequence Elements with SnapGene's Feature Database

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    Blog Post
    ...place the most important controls front and center, and make specialized controls available when needed....sequence and display frequently used genes and control elements. Development of this tool required creating... DNA sequence has changed. Identifying plasmid control elements Coding sequence features are relatively...relatively straightforward to define, but for control elements such as promoters and transcription terminators...obvious. We found that the annotated boundaries for control elements in commercial plasmids were inconsistent...how we use SnapGene during the plasmid quality control process Check out full plasmid sequencing at Addgene...

  10. Technique: Probe Phage Genomes for Host Binding Proteins

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    Blog Post
    ...or Salmonella, or any cells that bind strongly to nitrocellulose, over-incubation of plates can lead to...forcing DNA through a small opening using compressed nitrogen gas; this shears DNA at random locations and allows...containers that can easily be attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods...cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed to IPTG in order to induce...release recombinant proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity...First, cut out 20 Petri dish-sized circles of nitrocellulose membrane and press each gently down onto a ... helpful to spot this protein directly onto nitrocellulose and carry out the steps from blocking the membrane...

  11. Generating Mouse Models Using CRISPR/Cas9

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    Blog Post
    ...1980s, gene targeting technology was invented to introduce specific changes into the mouse genome. With these... early technologies, a researcher would first introduce a mutation into a mouse embryonic stem cell (ESC...chromatogram showing the telltale signs of a mutation introduced by CRISPR. The sequence started clean and then...handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant females, a technique...gRNA, Cas9, and donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  ...targeting. CRISPR tools were recently used to introduce a 25 kb human gene into the mouse genome. However...Goodwin LO, Morgan JL, Juan WC, Roca X, Ong ST, Bergstrom DE (2018) Viable Mice with Extensive Gene Humanization...

  12. Plasmids 101: Restriction Cloning

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    Blog Post
    ...on the left, your digested DNA (and undigested controls) are loaded at the top of the gel in wells positioned... uncut sample of each plasmid to help with troubleshooting if your digests don’t look as expected. Once...to insert. It is critical to set up a negative control ligation reaction with no insert DNA added. This...much less total DNA (<1ng) or if you are having trouble getting colonies, you might want to use higher ...to be very large (>10kb) you might want to use electro-competent cells instead of the more common chemically-competent...minimum, two transformations after a ligation: 1. Control Transformation containing the ligation mixture ...see any colonies, you should conduct a positive control to ensure that your transformation worked. You ...

  13. New and Upcoming Viral Vectors - September 2019

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    Blog Post
    ...will continue to add more controls, and have recently released these controls: pAAV-hSyn-DIO-EGFP (50457...sensors Optogenetics Recombinases And more! Control AAV For many AAV experiments, we recommend validating...aliquots in AAV1, AAV2, AAV5, AAV8, AAV9, and AAV Retrograde. You can also request the specific serotypes ...entire collection! We are expanding our entire control AAV inventory to give scientists access to a wide...AAV5 and 112677-AAVrg): The EF1a promoter directs strong, ubiquitous expression. This is a Cre-dependent... Email us or check our website to access them! Controls pAAV-hSyn-hChR2(H134R)-EYFP (26973-AAVrg) pAAV-hSyn-hChR2...

  14. Addgene-Seeding Labs plasmid grant round 3: microbial drug resistance and biofilms, antidiabetic plant components, visualizing brain injury - and a glowing robot fish

    Type
    Blog Post
    ...etiopathomechanisms of ischemic stroke, the most common type of stroke and the third leading cause of ...-Seeding Labs plasmid grant we are excited to introduce the 5 awardees and their research. These researchers... is necessary to improve tuberculosis care and control. In her current research, Sheen aims to identify...taraxacifolia (Willd), an annual herb of Western Tropical Africa typically known as wild lettuce. Aside ...of death and disability worldwide. An ischemic stroke occurs when arteries leading to the brain become ...of interesting combinations: biomedicine and astrobiology. During the last 11 years he has studied various...

  15. Antibodies 101: Isotypes

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    Blog Post
    ...you can select an appropriate isotype control. An isotype control has the same constant heavy chain domain...heavy-chain constant region (see Antibodies 101: Introduction to Antibodies for a refresher). There are five...molecules are recruited by the antibody to help destroy and remove a pathogen. Different isotypes also ...doesn’t bind your antigen of interest. Isotype controls are used to make sure the staining you see is ... than nonspecific background staining. Isotype controls are used in antibody-based applications including...techniques that you can use antibodies for in our Intro to Antibodies post Check out our Antibodies 101:...

  16. Viral Vectors 101: Biosensors

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    Blog Post
    ...The calcium is strong here….I can sense it….sound like a plot to an biology scifi movie? Or maybe it’...neurotransmitter, such as norepinephrine, or an electrolyte such as calcium. This varied component, which...For one, many viral platforms, such as AAV or retrovirus, can either permanently integrate and/or express...serotypes can be used to affect or determine viral tropism—the type of cells the viral particles infect. This... sensor, whether you choose lentivirus, gamma retrovirus, or AAV as your vehicle, we’ve got you covered...Resources References  Bhalla N., Jolly P., et al. Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60... Susanna Stroik...

  17. 15 Years of Addgene: The Top 15 Plasmids

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    Blog Post
    ...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and tet...pMD2.G. Find psPAX2. pMD2.G - Also from the Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid...envelope protein for producing lentiviral and MuLV retroviral particles. It can be used with packaging plasmids...as pCMV-dR8.2 dvpr (lentiviral) or pUMVC (MuLV retroviral). Fun fact: published in 2003, this plasmid predates...generation lentiviral packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used...generation lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It also...shRNA. This scrambled shRNA is used as a negative control for mammalian RNAi experiments.  Find the scrambled...

  18. CRISPR 101: Non-Homologous End Joining

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    Blog Post
    ...consequently the principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors are...ends tends to be the point where mutations are introduced. Repair of Cas9-induced breaks by NHEJ As illustrated...microhomologies”). NHEJ doesn’t obligatorily introduce indels. Given the end structure of the Cas9 DSB...hundreds of base pairs or more can simultaneously introduce a pair of chromosome breaks, and could result ...single strand break; when two such breaks are introduced near each other, in opposite strands. This “double...j.cell.2013.08.021 Waters CA, Strande NT, Pryor JM, Strom CN, Mieczkowski P, Burkhalter MD, Oh S, Qaqish BF...Video] Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens        ...

  19. Antibodies 101: Validation

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    Blog Post
    ...that antibody works as expected, you will see a strong antibody signal in the retinal sample, but little...you see a similar antibody signal between your control and your knock-out sample, you should be concerned...antibody binding in the transfected cells but not in control cells. But (there is always a ‘but’), you should...encoding this protein, which we will call Negative Control Gene (NCG) (A). Then you lyse the cells and run... your antibody (B). Your results show a lovely, strong band from the cells transfected with your protein...well as a third unrelated antibody as a negative control (A). The two antibodies to your protein of interest...sample using the antibody and then uses mass spectrometry to identify the captured proteins. If the antibody...

  20. When Fidelity Matters: A frank discussion about ligase fidelity

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    Blog Post
    ...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents...circularization of dsDNA (6). Less commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq...identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method allowed screening...gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology 45.6...PMC1933048. 16. Cheng, Changming, et al. "Electrochemiluminescent assay for detection of extremely rare mutations...mixed population of excess wild‐type DNA." Electrophoresis 34.9-10 (2013): 1415-1422. PubMed PMID: 23463388... PMID: 17466627. 25. Luo, Jianying, Donald E. Bergstrom, and Francis Barany. "Improving the fidelity of...
Showing: 261 - 280 of 916 results