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We narrowed to 881 results for: URE

Showing: 161 - 180 of 881 results

  1. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    Published
    Nov. 3, 2020, 2:15 p.m.
    ...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...

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

    Type
    Blog Post
    Published
    Dec. 16, 2015, 3:30 p.m.
    ... Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), ...Interactions between the target strand and the gRNA (see figure below) Because off-target sequences have less ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to ...improve the specificity of Cas9 derived from S. aureus. This means you can likely apply similar rational...of genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers..., Chen et al. 2017 used single molecule FRET measurements to determine which portions of Cas9 might be...HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With their enhanced...

  3. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in...plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters...also available as ready-to-use lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters...LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC...silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted ...period, as this mark was faithfully propagated in culture and in vivo. In certain cases, epigenetic modifiers...acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038...

  4. A Control for All Seasons

    Type
    Blog Post
    Published
    Oct. 5, 2023, 1:15 p.m.
    ...experiments require positive and negative controls to ensure proper interpretation of results. A positive control... to compare the experimental group.     Figure 1: A, Most biological experiments will benefit ...group and C, technical replicates or repeated measurements will increase the power of the results. In this...experiment. Technical replicates are repeated measurements of the same sample and are included to assess...long as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should...molecules through the interactions of other antibody structures, such as the Fc region. Fc-mediated binding interactions...as with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations...

  5. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    Published
    Jan. 10, 2023, 2:15 p.m.
    ...bacterial culture into the late stationary phase. Growing the culture at a lower temperature such as 30...  Figure 3. Summary of plasmid multimerization troubleshooting tips. Portions of this figure were ... More research is ongoing to determine plasmid features that affect the frequency of multimerization. .... Theoretically, as long as all the plasmid features remain intact, plasmid multimers function just as...recA-independent mechanisms. Avoid overgrowing the bacterial culture Another way to prevent plasmid multimerization ...optimizing the incubation time can help maintain cultures in log phase growth. In a study by Williams et...plasmid can exist in a bacterial population as a mixture of monomers and various forms of multimers (Summers...

  6. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...polysaccharide structures, many of which are easily varied by the cell and whose structures are just beginning...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell... for by manipulating the nebulization time and pressure. Nebulization units, which can be purchased cheaply...attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to subject your membrane...

  7. Finding Your Perfect Job After University

    Type
    Blog Post
    Published
    Jan. 12, 2016, 3:30 p.m.
    ...everything was more structured and controlled. I worked from Standard Operating Procedures (SOP’s) and every...perfect duration for me because it was sufficient to figure out if my image of lab work lived up to reality..., but in research you also experience a lot of failure and problem solving. I believe that is something...they never really prepare you for the amount of failure and problem solving scientists experience during...in ways that people from another background or culture could understand. I did, however, find it very ...learn how to cope with a different climate and culture, and also needed to be resourceful and adapt. By...doing experiments  without the constant fear of failure because I knew the tests worked and would produce...

  8. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    Published
    June 25, 2020, 1:15 p.m.
    ...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst... member of the Quality Control team, I help make sure the plasmids that go out our doors are correct. ...minor, it’s time for me to start considering my future path. Talking to Addgenies has helped me get a ...concepts that are heard often during undergrad. Lauren Flynn is the Addgenie behind our "Meet an Addgenie...years ago at a CRISPR meeting.Read his Instagram feature here. You also don’t have to go back to school...that if you don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize...with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya Vedantam...

  9. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ...through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed...difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a... to knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert...for targeting constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and...-eGFP-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed... and the targeted exon in the gene of interest (Figure 4). When recombination occurs, the gene still functions...cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed allele...

  10. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...long dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire...involved in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing... modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...that facilitates the formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi...greatest advantage of RNAi is the straightforward nature of the technique—all that is required dsRNA complementary...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be ...

  11. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ...halt degradative processes while maintaining the structure of your samples, the relationships between cellular...that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...cross-linking, but it doesn’t preserve sample structure quite as well as a cross-linker. This is because...fixation with these chemicals, which impacts sample structure and could also cause your target to be washed ...Furthermore, these chemicals can also alter the structure of proteins, which could disrupt your target epitope...good thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...dehydrates the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear...

  12. MXS Chaining

    Type
    Blog Post
    Published
    Feb. 7, 2017, 3:30 p.m.
    ...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the...various mammalian promoters), the same cloning procedure can be used to incorporate each element into the...These fluorescent reporters can be used to detect structure and protein localization, as well as cellular ...with MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...fluorophore, and thus enabled labeling of particular structures (Table 1). Each construct was flanked with a ...final construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling...Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation/emission...

  13. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...bacterial recombination pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr ...hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol...cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid...design for each type of modification is shown in Figure 2, and is discussed in more detail below. The main... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple...

  14. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...vectors effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used ... you can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral particle, which...   Figure 3: The life cycle of a retrovirus. Image courtesy of Jeremy Luban, MD. Figure 3 depicts...vectors correctly. A solid understanding of the structure of a wild-type viral particle — and of the normal...depicts the viral life cycle. One should ponder this figure often, especially the three steps from the 12 o...

  15. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 ...domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split" cas9...for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs ...light-mediated subcellular localization in mammalian cell culture and reversible control of signalling pathways. ... off and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant....g. the ER) and a reporter protein of interest (Figure A). Franck Perez’s lab has engineered hook proteins...

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

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...Zhang, a Ph.D. candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose... now take place. After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA...other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new...us to update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition...DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...

  17. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    Published
    Sept. 21, 2017, 2:07 p.m.
    ...toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes...genes that fell into 3 categories based on the architecture of the CRISPR protein in question: C2c1, C2c2... referred to as Cas13a) is unique in terms of structure and function, and will therefore be the focus ...cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology to the most commonly...CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of...applications of Cas13a given what we know about its structure and function? For starters, Cas13a can be used...2017). This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika...

  18. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ...required longer exposure time than its protein equivalent and was less stable due its RNA nature, it paved ...including: Environments with higher temperatures, since proteins can denature while aptamers are more stable...form the base of the stem in an RNA stem-loop structure (see Fig. 1a). When the aptamer binds its ligand...laboratory took advantage of their deep knowledge of the nature of GFP fluorescence to create an improved fluorescent...fluorescent aptamer system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium... its biosensor capabilities (see Fig. 1b). The future of aptamer fluorophores The Jaffrey lab and others...

  19. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    Published
    Aug. 31, 2023, 1 p.m.
    .... These features enable the fibers to emit light for optogenetic purposes, measure temperature, study ... described, then measured electrophysiological potential afterwards. This measurement trended with expression... the fibers would need to be, as well as other features necessary for implantation in brain tissue. The...the wild west for fiber implants. Due to the soft nature of the intestines and the requirement for digestive...multiple channels and can also record tissue temperature proximal to the implanted fibers. As important...Alternatively, the system could be used to deliver or measure new variables. The sky is really the limit for ...wireless modulation of gut and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587...

  20. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

    Type
    Blog Post
    Published
    Sept. 19, 2017, 1:20 p.m.
    ...experimental settings: HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing...just 1 minute of exposure. MG-2I-dL5** experienced minor bleaching after longer exposure times. Additionally... fluorogen that can be used to ablate cells in culture and in vivo.  Read on to learn more about this ...exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient for...mCer3 cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...light. They found that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the...genetically targetable near-infrared photosensitizer. Nature Methods,13(3), 263-268. doi:10.1038/nmeth.3735....
Showing: 161 - 180 of 881 results