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...Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed...

...process is called α-complementation. The system described above was put to practical use in the following...α-complementation cloning MCS): pGEM-T, pUC18 and pUC19, and pBluescript are a few common vectors. Blue-white screening...dependent on certain media components, and are rescued by genes supplied on the transformed plasmid. Such...complement auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources on the Addgene Blog...

...locus-specific knock-in of FKBP12F36V. Detailed protocols describing the use of these plasmids are available here....al. and Weintraub et al.), chemical and genetic rescue experiments (Nabet et al.), proteomics (Nabet et...degradation, and lead author on the recent paper describing the dTAG technology. Follow him on twitter @behnamnabet... 29581585. This reference contains the full description of the dTAG system. The dTAG system was employed...

...includes overview information, key dates, funding description, research plan, award information, eligibility...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...project are clearly stated and align with the description of the RFA and funding institution. Emphasizing...

...works best with western blots that use chemiluminescent or fluorescent detection, like horseradish peroxidase...that involves adding fresh chemiluminescent substrate or imaging your fluorescent probe. Then, if you don...

...cloning, the Haynes lab’s Protocols.io protocol describes an elegant system that allows for the assembly...implications for the fusion protein effectivity), and a fluorescent protein tag for visualization (Fig. 1b). While...catalytically inactive dCas9) to modify chromatin. A fluorescent protein can still be included for visualization...quantify protein degradation the Ward lab used fluorescent proteins (FPs) fused to the AID* (minimal 44 ...

...Green or Taqman for signaling. Sybr Green is a fluorescent dye that works by binding to all double-stranded...while Taqman uses an intercalating dye, with a fluorescent probe and quencher to quantify specific amplicons...materials, and is not particularly sensitive. The fluorescent signal can be affected by AT-rich DNA and the... Sybr Green chemistry. Blue arrows represent fluorescent signaling in the presence of dsDNA. Image courtesy...

...sequences for several different fluorescent proteins. Visualizing fluorescence can be challenging in plants...because of the auto-fluorescence of chlorophyll, so we looked at newer fluorescent proteins that get around...

...made news in March with a publication in Science describing the development of “transcriptors,” transistor-like... and observed that we humans find it useful to describe and interact with the world using words like OR...The future of synthetic biology Addgene: You've described the pieces in these kits as the beginnings of ...regulation of gene expression including growth or fluorescence. There are a few examples of engineered RNA ...

... the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence is present...resulting in activation of the fluorophore. The fluorescent signal can be used as an indicator to determine... the collateral RNA cleavage response is well described in bacterial cells, LwaCas13a does not display...programmable RNA-binding protein, creating the fluorescent fusion protein dLwaCas13a-NF for in vivo RNA ...

...LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes...slow-growing protein fiber in the cytoplasm and can be fluorescently labeled. Using lentiviral vectors to stably ...different promoters, specific time points appear as fluorescent bands in the fiber, resembling a “ticker tape... treated with a phorbol ester activator gave fluorescent signal in the ticker tape from cFos::GFP-iPAK4...

...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the absence of Cre....) are generated from lentiviral preparations (described above) that are subject to a concentration step...10.1089/hgtb.2013.131 Wang, Y., Bergelson, S., & Feschenko, M. (2018). Determination of Lentiviral Infectious...

...are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used to study...without signs of toxicity or burden relative to a fluorescent protein control. We also added the toxin-antitoxin...mammalian cells. Science 365, 1469–1475 (2019). Maresca, D. et al. Nonlinear ultrasound imaging of nanoscale...

...CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems. The original pCas... that same year. Her research is focused on the escape mechanism of genome editing in microorganisms which...Yang J, Yang S (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl ...system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochimica et Biophysica Sinica. https...

...genotype to phenotype studies New organismal systems described at this meeting were extraordinarily diverse. ... manipulated by injected RNA and can express fluorescent proteins. This lab has created an automated tank...transgenic animals. They might be immortal. Manihot esculenta (Blake Meyers) Cassava is the fifth most grown... process is expensive. They are using a cool fluorescent indicator system to rapidly identify recombinants...

...generally not true. Because of the variations in fluorescent signals, and how the machine reads/interprets...light that “scatters” past the cell, instead of fluorescent markers. There are various types of SSC and FSC...which is comprised of antibodies conjugated to fluorescent markers and/or cellular dyes and stains. These...anti-CD14 and anti-HLA-DR antibodies conjugated to fluorescent markers are used to identify monocytes (upper...

...If you’re new to Rett Syndrome, this blog post describes the history of Rett Syndrome research, the role...MECP2 in Rett Syndrome Rett Syndrome was first described in 1966 by Austrian physician, Andreas Rett, who... a portion of the MECP2 protein is necessary to rescue the neurological defects of Rett syndrome (Tillotson...Cobb SR, Bird A (2017) Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature...

...insert into RNA. In practice, the term "promoter" describes the combination of the promoter (RNA polymerase...   Primarily used for RNA     transcript     Description Expression Additional considerations CMV General...promoters     Promoter     Primarily used for Description Expression Additional considerations T7 in ...identify the right tissue-specific promoters as described by researchers at the NIH here.  Note: A. Max...

... I thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low...Plasmid #52962) was a gift from Feng Zhang and is described in Improved vectors and genome-wide libraries ...was a gift from John Doench & David Root and is described in Rational design of highly active sgRNAs for...were gifts from John Doench & David Root and are described in Optimized sgRNA design to maximize activity...

...Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase." Journal of bacteriology 173.16...PMC1933048. 16. Cheng, Changming, et al. "Electrochemiluminescent assay for detection of extremely rare mutations... probe DNA-immobilized magnetic beads and chemiluminescence detection." Analytical Sciences 29.7 (2013..."Optimization of a ligase detection reaction-fluorescent microsphere assay for characterization of resistance-mediating...