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...bacteria CRMAGE system combines CRISPR and recombineering-based MAGE The Nielsen lab’s CRMAGE system is...Pedersen LE, Sommer MOA, Nielsen AT (2016) CRMAGE: CRISPR Optimized MAGE Recombineering. Sci Rep 6: . https:/...method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology...self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more efficient than traditional recombineering...simultaneously is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours...

...depends on both the biological system as well as the imaging method, we recommend basing your selection on a...application. The three key properties for live cell imaging that will be treated in this series of posts are...experimental conditions that are related to the imaging strategy and the sample. Practical brightness A...mammalian cells is to tag an endogenous gene. By imaging cells (or tissues) that produce an endogenous protein... be used to verify performance under different imaging conditions (e.g. different emission filters) or...Choosing Your Fluorescent Proteins for Multicolor Imaging Learn about Fluorescent Protein Biosensors Additional...

...this post: Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized genetic screening...and editing Anti-FLAG Frankenbodies for modular imaging of cellular targets by: Mike Lacy Adding to their...a variety of applications, including live-cell imaging, single-molecule tracking, immunolabeling of fixed... be streamlined into a single array for Cas12a. Image from DeWeirdt, et al. 2021 To validate this ...expressed and coexpressed proteins run as ladders. Image from Santilli, et al. 2021 Plasmids are used...of CasMINI for genome engineering applications. Image from Xiaoshu, et. al. 2021.   Created by the...

...Metal spatula Razor blade Plastic tray Gel imaging system ImageJ or similar software Reagents Purified recombinant...the sink. Take a brightfield image of the gel with an appropriate imaging system. Figure 1 Recombinant... QC and would not be used. Section 3: Image Analysis Use ImageJ (Link opens in a new window) or a similar...bands to the overall lane. Import the gel image into ImageJ. Select File . Select Open . Choose the location...location of the file to open. Change the image type to 8-bit . Select Image . Select Type . Choose 8-bit . Determine... using ready-to-use biosafe Coomassie stain and ImageJ software. Protocols...250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing...

...of the antibody binding “distinct imaged pattern.” The distinct imaged pattern should align with what the...contains secondary antibodies, allowing us to only image autofluorescence i.e., background noise. We then...and not from the background noise. If the sample images similarly to the negative control, that informs...on the antibody signal’s location. If a distinct imaged pattern does NOT align with the atlases/literature... own analysis of the background noise, distinct imaged pattern, and exposure times. To test the antibody...

...antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019.   Competitive...antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019.       Direct ...antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019.   Indirect ELISA...antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019.   Detecting the...this line to calculate the protein concentration. Image from Jagarlamudi et al., 2015.   The maximum...Urbaniak, S. et al. Review: immunoassays in DNA damage and instability detection. Cell. Mol. Life Sci....

...AC, BC, and ABC). Nielsen Lab E. coli CRMAGE Plasmids: The CRMAGE system is a fast, multiplexable method...12. Ronda, Carlotta, et al. (2016). "CRMAGE: CRISPR Optimized MAGE Recombineering." Sci Rep. 6:19452. PubMed...method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology...self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more efficient than traditional recombineering...simultaneously is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours...

...sea anemone's red color can be used for mimicry. Image: Bernard Spragg. Advantages of using chromoproteins...Chromoprotein expression from bacteria on agar plate. Image: Liljeruhm et al., 2018. For each chromoprotein... plasmids expressing amilCP and tsPurple genes. Image: Lazarus et al., 2019. One such lab is the Waldor...chromoprotein is expressed and gives the cell a magenta (amilCP) or purple (tsPurple) color. Though the... used as a FRET acceptor to mRuby2 or mScarlet. Image: Murakoshi et al., 2019. ShadowR is an orange...developed as an acceptor for fluorescence lifetime imaging microscopy-based Förster resonance energy transfer...

...Choosing the best PE6 variant for your application. Image reused form Doman and Pandey et al., 2023 under ...fusion and targeting components are interchangeable. Image from Strebinger et al., 2023, reused under CC-BY... trafficked from the “source” to “destination”. Images adapted from Lee and Cheah et al. 2023 bioRxiv ...GFP-iPAK4 within an hour. Analyzing the ticker tape images could recover transcriptional histories, accurate...labeled with HaloTag-iPAK4. Scale bar 10 µm. C) Images of iPAK4 fibers in cultured rat hippocampal neurons...induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted from Lin et al., 2023 bioRxiv, under CC-BY-NC...

...studies that involve surgery or imaging.  Rattus norvegicus. Image from Staffan Vilcans. Rat physiology...Idalia & Bernardo, 2017).  Escherichia coli. Image from NIAID. E. coli is often a part of the “molecular...mexicanum) Ambystoma mexicanum, aka the axolotl. Image from AJC1. The Mexican axolotl is one of the ...biomedical research.  Xenopus, aka African frog. Image from Holger Krisp. The frog genome has high synteny...adaptations.  Arabidopsis thaliana flowers. Image from BlueRidgeKitties. If you are working with...

...immunofluorescence and indirect immunofluorescence work. Image created with BioRender.com   Indirect IF is ...access their target and that you will be able to image them in the end. Again, the methods you use will...time for this step as well. Preservation and Imaging Similar to Sample Preparation, this step can ...procedure with key considerations about each step. Image created with BioRender.com   The first couple.... the spectral properties match your available imaging instruments and the spectral properties do not ... pressing research questions, but also stunning images (am I the only one with IF results as artwork at...

...vivo. Merged green and red channel fluorescence images of C2C12 cells stably expressing pTRE-Tight-MitoTimer...pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence microscope ...organelles with older (yellow to orange) protein. Image courtesy of Roberta Gottlieb. Laker et al., ...exhibit superior performance over APEX for both EM imaging and proximity-dependent proteomic mapping with ...which can be fused to proteins of interest for EM imaging, or targeted to subcellular regions or protein ...40.  Next-Gen Brainbow toolkit for neuronal imaging Joshua Sanes and his team at the Center for Brain...Brainbow toolkit for high-resolution fluorescent imaging of individual neurons. The technology generates...

...variety of neuronal imaging applications, including confocal and widefield imaging, two-photon microscopy...Figure 3: PinkyCaMP and FRCaMPi in use. A) Confocal image of CA2 neurons in a mouse brain slice expressing.... (2024) under CC BY-NC-ND license. B) Confocal images showing fore-, mid-, and hindbrain regions of live... red fluorescent calcium indicator for in vivo imaging of neuronal populations at single-cell resolution...liquid transfer, incubate at room temperature, and image with smartphones. The Qi lab also developed an algorithm...

...PsMOrange and mEos add another layer of complexity to imaging, as they allow one to monitor compartment/protein...subsequently confirmed that purified GFP could be imaged in the green state after excitation at 488 nm, ...the red state after excitation at 561 nm. This imaging process worked well with the common GFP variants...spreads through the cytosol via cytoplasmic strands. Image from Sattarzadeh et al., Sci Rep. 2015 CC BY 4.0...variety of other fluorescent proteins for your imaging needs. If you need an FP refresher, see our FP ...

...fluorophores for imaging Lee S, et al. (2013) PLoS ONE 8(7): e67902. Many imaging studies rely on...which nearly all domains of life repair genomic damage, specifically double strand breaks. Researchers...recommend optimal FP combinations for use in yeast imaging, categorized by specific filter sets and experimental...tagging vectors for your single or multi-color imaging experiments. Interested in epitope tags? Others...

...level provided by SunTag is so high, cells may be imaged under lower illumination settings, lowering photobleaching...phototoxicity concerns and extending the potential imaging time for this technique. The NIH Common Fund has...based method is a great step forward in live cell imaging of genomic loci. Ma et al. anticipate that the ...plasmids from this publication at Addgene. Dynamic imaging of genomic loci in living human cells by an optimized...amplification in gene expression and fluorescence imaging. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, ...

... family of calcium sensors and can be used for imaging of small neuronal processes like dendrites and ...neurotransmitter in vertebrates, but methods for directly imaging GABA have been limited. Like calcium sensors, different...to monitor neuronal electrical activity through imaging. Since it’s illuminated by red/orange light, Archon1...resting fluorescence. It is useful for activity imaging of large populations of densely labeled neurons...resting fluorescence.  It is useful for activity imaging of large populations of densely-labeled neurons...

...in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  Wensheng Wei's lab at...indicator, GCaMP6f, in the Drosophila optic lobe. Image from Isaacman-Beck et al., 2019. Get started... stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. Find the GFP reporter...with the fluorescent protein fluorescing green. Image from Shin et al., 2019. Looking for an alternative...not cause structural deformation or photo-induced damage. These fusions can also function in high salt conditions...

...fiction. Two dueling magicians in the 1800s in England who help bring back actual magic (which had been lost...features a young female protagonist, some clever magic, and a satisfying conclusion? In this series, there...even more sinister. 1000 pages long. Incredibly imaginative, funny, bittersweet, and shocking at times."     ...ve read books across nearly every genre you can imagine: gothic, mystery, sci-fi, fantasy, pop-sci, true...

...external signals such as developmental cues and DNA damage. These signals activate a cascade of caspases, ...after proteolytic cleavage. Secondly, fluorescence imaging of living organisms can be challenging due to issues...array of multicolor reporters that will enable imaging of multiple proteases at once (Zhang et al., 2019...of a GFP-based fluorogenic caspase reporter for imaging apoptosis in vivo." Cell chemical biology 23.7 ... reporter by flipping a beta strand of GFP for imaging apoptosis in animals." Journal of the American ...