...or fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of ...Non-destructive methods, most popularly with a fluorescent reporter, directly couple silencing of your gene ...that contains a target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore...readily observable phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed...identified as undergoing RNAi through the fluorescent reporter and manually extracted. This process is ...of using a fluorescent output to monitor RNAi, we engineered a positive selection reporter whereby survival...gene target to silencing of the reporter. This is achieved at the level of the DNA construct, where your ...

...thaw cycles. Depending on the researcher and the type of virus, there have been varied reports of titer losses...should appear fluorescent if they were infected. While there may be some low-level fluorescent protein or...Transduction efficiency depends on viral titer Both of these can be increased by concentrating a viral...1. Viral vectors undergo DNA rearrangements. Depending on the composition of the viral vector and the...virus. Alternatively, if the virus encodes a fluorescent protein, the packaging cells could be examined..."Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption... prior to their transduction has been shown to increase AAV transduction efficiency by approximately 15...

...knowledge of the nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that...oligonucleotides in fluorescent visualization techniques for decades, most notably for fluorescent in situ hybridization...applications including aptamer based fluorescent biosensors. Aptabeacons Fluorescence resonance energy transfer ...aptamers fluoresce until they bind to the appropriate ligand, but relying on a lack of fluorescence for readout...applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing with veggies) Though the...aptamer that would bind the fluorogen and increase its fluorescence, essentially mimicking GFP.  Though the...highly customizable in terms of how they actually “report” as biosensors. Making aptamers glow The flexibility...

...C-terminus of endogenous proteins. The PITCh system depends on MMEJ (microhomology-mediated end-joining), an...vector assembly, and the remaining 5 plasmids include Cre and FLP expression vectors, a general cloning plasmid...with the following article: Natsume, et al. Cell Reports 2016 Förstemann Drosophila Cell Tagging System ... the first publicly available collection of fluorescently tagged, human, induced pluripotent stem cell..._CEBPA_1 PX458_CEBPA_2 CREB1 Human FLAG pFETCh_CREB1 PX458_CREB_1 PX458_CREB_2 TGIF2 Human FLAG pFETCh_TGIF2...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...genome modification. This system can be used to create C- and N-terminal epitope tags. The plasmids in...

... the fluorescent protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase...). Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized...of the fluorescent proteins used in the system. One such system is Brainbow 2.0, which uses Cre to trigger...default, they are not fluorescent. Instead, they incorporate a truncated, non-fluorescent form of YFP as the...is necessary to excise the non-fluorescent YFP and activate fluorescence of the downstream OFP, GFP, or...combination of all of the fluorescent proteins from the injected construct. The fluorescent protein expressed ...developing fish via Cre protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier...

...additions, including our first far-red fluorescent marker, a Flp-dependent channelrhodopsin in the retrograde...help, with over 100 user-submitted viral vector reports. And don't forget to use the link below to keep...Biosensor GENIE Project New serotype pENN.AAV.CMVs.Pl.Cre.rBG AAV5 Recombinase James M. Wilson New serotype...

...such as fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy... have a variety of fluorescent light sources. When interrogated with a fluorescent light source, a cell...photons varies from cell to cell depending on the level of fluorescence. A cell expressing high levels ...proteins tag your protein of interest with a fluorescent reporter on the N- or C-terminus of the protein. One...conjugated fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by ...laser and interrogated with various visible and fluorescent light sources that allow users to assess a cell... cells in a population in a procedure termed fluorescence activated cell sorting or FACS (2). By analyzing...

...years — a green fluorescent protein, a red fluorescent protein, a fancy new green fluorescent protein, some...new-and-improved fluorescent protein you want to try, just swap in a new fluorescent ligand and see the...purification tags, another version with that same red fluorescent protein somebody made because they forgot it ...of chemical dyes provide many advantages over fluorescent proteins, opening up new applications.   ... self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from Roger Tsien’s lab ...dihydrofolate reductase (eDHFR) could be labeled with fluorescent derivatives of its substrate trimpethoprim (TMP-tag...collection. CLIP-tag: Also in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag, which reacts...

...FAST system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors...the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated that...while transcription of the N-terminus is light-dependent (Yu et al., 2020). To express the N-Cas9, Haifeng...protein BldD to the nucleus. To couple this light-dependent activity to transcription, they fused BldD to ...of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa) (Zhou et al., 2018). pdDronpa... flanking the DNA-binding cleft of Cas9, the fluorescent domains dimerize and prevent DNA binding in the...chromatin architecture (Hoffman et al., 2020). Fluorescent dCas9 proteins can be used to identify specific...

...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such...dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate...capture reagent may be an antigen or an antibody depending on how the assay is set up. Then they block the...peroxidase (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we ...we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users provide... sandwich and competitive. In a direct ELISA a reporter enzyme conjugated antibody binds to antigen coated...antibody first binds to the antigen and then a reporter enzyme conjugated secondary antibody binds to ...

...may be a plasmid that constitutively expresses a reporter protein (e.g., GFP or luciferase) that is either... well of your cells. Regardless, the amount of reporter protein activity correlates to both the amount...successfully and expressing the protein. For example, fluorescence microscopy images resulting from our experiment...was not successful due to the absence of GFP fluorescence from the living, viable cells. This result presents...produces the expected effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid...These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...Plasmid A is the decreased expression of Gene X. Ergo the positive control(s) should decrease the expression...

... is the permanent fluorescent tag portion of Rosella: it will emit red fluorescence regardless of its ...the fluorescent properties of Rosella.   Table 2: Effect of Localization on Rosella’s fluorescence emission...green fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks ...Devenish, R. J. (2008). Rosella: A fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and...mammalian cells using a single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed...Photoconversion Light Sheet Fluorescence Microscopy Additional Resources on Addgene.org Fluorescent Protein Plasmids...Plasmids and Resources Fluorescent Protein Guide: Biosensors Keima, another fluorescent pH-biosensor for studying...

...good reporter genes There are many great reporter genes available and which one you choose depends on your...the T-DNA region with our gene of interest or a reporter gene so that the transformation and gene expression...different treatments on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase...histochemical GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence...half-lives then GUS and are therefore good temporal reporters to measure promoter activity, for example. Useful...tNOS & pSB166 - pL2_pSB90_tMAS::rLUC-I::pMAS. Fluorescent proteins (FP, e.g. GFP) can be used in many ways...

...neomycin, puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors ...SfiI restriction sites with different overhangs depending upon whether they are on the 3’ or 5’ end of the...region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new tool kit allows... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...transposase gains its name from the fact that it was recreated from a defective transposase found in fish. This...transposases. Improvements have been made since then by increasing the consensus between a greater number of extinct...primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB tranposase variants for hyperactivity ...

...Passive reference dyes are inert fluorescent dyes that are not dependent on amplification of the DNA; they...step. Scorpion chemistry in particular has been reported to work well in the Lightcycler while being relatively...fluorogenic primers Low Single self-quenching fluorescent molecule connected to a primer  No Yes ... Agarose gel Sunrise primers   Low Reporter and quencher on hairpin structure included in primer...much as sixfold variation in expression levels reported (Biassoni, 2014), so you’ll want to ensure that... It is possible to multiplex qPCR when using fluorescent probe-based chemistry - theoretically, up to ... Tip🔥 The ABI machines automatically record fluorescent data on all channels - helpful if you forget ...

... of Cre-dependent mouse lines for overexpressing genes within discrete cell populations makes Cre-dependent...the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids used to express CRE under...importantly, the expression of this construct is Cre-dependent, enabling spatiotemporal control over its expression...Constitutive or inducible Cre recombinases can be introduced by AAV or by mating with Cre-expressing mice. ... many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired with ... used to express Cre under neuron-specific promoters, including calmodulin-dependent kinase II alpha (...The pooled nature of this experiment requires fluorescence activated cell sorting (FACS) to isolate differentiated...

... Using an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37...combinatorial library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid set 3000 Series...initiation and instead makes initiation heavily dependent upon the core of a Shine-Delgarno sequence found...strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators.... coli, Bacillus subtilis, or Pichia pastoris depending upon your specific protein folding needs. The...fusions. Some applications of PUF protein fusions reported in the literature include RNA detection, RNA cleavage...greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of plasmids...

...there is no fluorescence background resulting from nonspecific donor labeling. Fluorescent proteins (FPs...incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (...Experiments Beware of Fluorescent Protein Aggregation Brush up on Your Fluorescent Protein Basics Resources...Browse Empty Plasmids to Fluorescent Protein Fusions Check out Our Fluorescent Protein Guide ...variants have been created, thereby enabling a broad spectrum of donor fluorescence wavelengths for FRET...answers. What is FRET and what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical...probes are close together (typically 30-100 Å, depending on the probes). In fact, FRET can be used to derive...

... from one compound to the other. These biotin dependent enzymes are essential for processes such as gluconeogenesis...primary antibody respectively. Avidin bound to a reporter is then added to the reaction for easy detection... experiments. During detection, a biotinylated reporter enzyme that is pre-incubated with free avidin ...protein of interest leading to amplification of the reporter signal and easier detection. This strategy is ...Attaching polypeptides to a substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization...labels that can be added to an oligonucleotide depending on your downstream application. Biotin labelled...complex can bind to your biotinylated antibody. This creates a large meshwork of biotin-avidin complexes bound...

... how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible Cre, such...invert as long as Cre remains expressed; once Cre is removed from the system, the fluorescent protein whose... not fluoresce, but can be detected via immunostaining. This feature facilitates screening of Cre-negative...Cai et al. also created an Autobow construct containing both Cre and XFPs. Cre production drives recombination...continued utility of Cre-lox. Check out our previous blog post, Plasmids 101: Cre-lox, if you need a quick...three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP (membrane tethered cyan). Without Cre recombination...same. The first fluorescent protein in the construct, OFP (orange), is expressed when Cre is absent; stochastic...