... Scale bar, 2 μm. Inset shows comparison with standard confocal microscopy. Image adapted from (Bottanelli...Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007 Griffin, B. A., Adams, S. R., & ...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...black is the fusion tag or labeling system. Image adapted from Lavis, 2016, under CC-BY. Common self-...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible... conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ...based on your experiment. You can see from Figure 2 that determining where to place your gate based on...the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing a range of antibody...plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify... samples may allow for better interpretation. Priyamvada Prathima has a research background in protein...

...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ...

...of other Cas proteins found in the so-called class 2 CRISPR-Cas systems. There are 24 variants of the cas14...the presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific...Last updated Apr 17, 2020. Before being adapted by scientists to edit the genome of virtually any organisms...on this planet, CRISPR-Cas systems were merely adaptive immune systems that provide bacteria protection...

.... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

...specific control of activity. Read More CRISPR Class 2 C lustered R egularly I nterspaced S hort P alindromic...alindromic R epeat (CRISPR) systems, which form an adaptive immune system in bacteria, have been modified ...

...Homo line 2) or wild type (WT), two weeks after herbicide was applied to both plants. Adapted from Vu et... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET... B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY 4.0 license...(C) is indicated; red line is exponential fit. Adapted with permission from Hao and Lee et al., 2024. ...non-neurodegenerative diseases (Diabetes & cancer). Image adapted from Wu and Wang 2024 under a CC-BY 4.0 license...

...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

... 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and Anders S. Nilsson. "Adapting drug approval... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...moved from the University of Alberta in Edmonton, Canada, to the University of Georgia, USA) has developed...

... Figure 2: Strategy for assembling barcoded combinatorial gRNA libraries. (Adapted from Wong et ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...for assembling the barcoded gRNA library pool. (Adapted from Wong et al., 2016). With a list of gRNA...

... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice.../Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers... gene (although it has been quite successfully adapted for use in screening experiments as well). Therefore...fluorescence indicates a more successful gRNA design. Adapted from Mashiko et al., 2014. Not all gRNA’s are...chance of contamination with RNase leading to degradation that can set you back days. Working with plasmid...

...and a source of sdAbs (Figure 2) (Xu, et al., 2021).      Figure 2: Genetically modified mice offer... from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature 595:278–282. https://doi.org/10.1038...single-domain antibodies. Image created in BioRender and adapted from Xu, et al., 2021.   The single-domain ...

... systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein...and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA-7.10. It is ...deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with ...evolution of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme...additional mutation (V106W) into TadA-8e and the resulting ABE8e(TadA-8e V106W) was as efficient in on-target...codons within T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage...

...method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...phase   Table 2: A comparison of different qPCR analysis methods. Table adapted from Wong, 2018.  ...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design of primers...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674... the simplest CRISPR systems and the easiest to adapt to genome editing, but the introduction of type ...positions are indicated below each graph. Figure adapted from Kleinstiver et al., 2016. Until recently...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization of Adeno-Associated...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...however, the TOPO cloning technique has also be adapted for blunt end cloning. So how does TOPO cloning...

...phages but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip..., accessed 25 November 2013. Author: James Atmos (2). Many CRISPR/Cas moieties carry out these functions...originally utilized CRISPR/Cas as a powerful and adaptive defense strategy against life-threatening viruses...scientists to hypothesize that prokaryotes developed an adaptive immune system that utilized various cas genes ... of anti-CRISPR genes in phages The CRISPR/Cas adaptive immune system seems like the winning ticket to...