...Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing vectors at Addgene! Multiplexing...expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet),...), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid...plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more ...PMID: 25337876. PubMed Central PMCID: PMC4270925. Find plasmids from this paper at Addgene. 2. Kabadi,...PMID: 25122746. PubMed Central PMCID: PMC4231726. Find plasmids from this paper at Addgene. 3. Sakuma,...PMID: 24954249. PubMed Central PMCID: PMC4066266. Find plasmids from this paper at Addgene. 4. Albers,...

... many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous...process! As NHEJ is ineffective in bacteria, CRISPR-induced double stranded breaks (DSB) are lethal. Addgene...persists, Cas9 will cleave the gene of interest, inducing a lethal DSB.     This system is distinct from...expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet),...), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid...plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more ...Enterobacteriaceae, without the need for modification. This finding suggests that the system may be functional in most...

...This post was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta. If you...in real time PCR, each reaction partition is individually assessed for a positive or negative (digital...introduced how digital PCR works, let’s explain what kind of assays can be used to sniff out your desired ... through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous end joining...to generate translational reading frame-altering indels for “functional knockout” of a gene of interest...applications of precision genome editing, the remainder of this post will focus on assays to detect NHEJ... locus, as well as a reference probe designed to bind at an unedited site distal tothe predicted double...

...combination to find suitable antibodies for a target with very low expression, due to induced toxicity caused...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...time-consuming and expensive, it may be helpful to find an antibody that has already been validated for ...leukocytes. Where data is available from antibody-independent methods, like RNAseq or proteomics, it is possible...expression should have less antibodies labeling them. We find that cell tracker dyes are helpful in designing ....g. PMA or a specific cytokine) known to either induce or suppress the expression of a target of interest...

...antibody-specific antibodies bind to and recognize the constant region. Nonspecific binding, caused cell receptors... a different antigen; binding of each can be either simultaneous or independent of the other (Shahied,...that have the ability to specifically recognize and bind proteins. They’re used in a number of research and...final product. Because the constant (non-epitope binding) region is the first thing to go when downsizing...These reduced-in-size options often have reduced binding strength, half-lives, and/or specificity as well...of the variable region (the antibody region that binds to the antigen) from existing antibodies. Each cleaved...make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker, resulting...

...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids Topics...gRNA and making strategic mutations in the DNA binding cavity of Cas12f. The CasMINI system is less than...active nuclease.  Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing with...to VP64. Read the paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing...AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   Selective endogenous encapsidation... The retroviral-like protein PEG10 specifically binds its own mRNA to facilitate its packaging into virus-like... other gene delivery. Read the paper in Science Find the SEND plasmids    Figure 1: SEND combines...

...’s antigen binding function but not the Fc region (more on when that might be later). Find scFv plasmids... region (or the “Fc”) and two Fragment Antigen Binding regions (or “Fabs”). The Fc region is the “tail...secondary antibody you should use when performing indirect immunoassays. The Fab regions, on the other hand...Fabs are the regions of the antibody that actually bind antigens.      Figure 1: IgG antibody structure...available different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced...hinge region disulfide bonds, resulting in two independent Fab fragments with small tails of the hinge region...above the hinge-region, which results in two independent Fab fragments. Size: ~50 kDa. Fv - These fragments...

.../conditions can change binding ability – i.e., the antibody can no longer bind to the target). Unreliable...specific binding and has a strong signal. We call the unique pattern of the antibody binding “distinct...spent hours sifting through comments and papers to find the most reliable (and cost-effective) antibody ...causes of this unreliability: cross-reactivity (binds to non-target proteins), variability (separate batches...goldilocks antibody is highly specific (minimal binding to off-target molecules), reliable (wouldn’t perform... choosing which antibodies to screen, we kept in mind our own lab’s purposes. The Wu lab mostly uses whole-mount...Strength and accuracy The strength is based on binding specificity and exposure time. We first have a ...

...through binding of the molecular species. Administration of these dyes results in direct binding between...fluorescent ligand when designing an experiment. You may find yourself defaulting to the most popular or familiar...experiment, including the accuracy and efficacy of findings. If you’re struggling to select the right fluorescent...with excitation/emission peak at 495/517 nm. FITC binds to an amine on a protein, enabling the labeling ...antibody, whereby excitation of blue light will indicate where FITC is bound through green light emission...dye-conjugation affects protein function and its propensity to bind cellular components. Some dyes target specific organelles...flexibility due to the affinity tag they typically bind to. Stability of fluorescence: For applications ...

...Visualization Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent...introduction to fluorescent biosensors, but keep in mind that there is a lot of variety in how biosensors...(CaM) and the chicken myosin kinase calmodulin binding region RS20. When calcium is absent, the construct...construct does not fluoresce; when present, calcium induces CaM/RS20 interaction, thereby changing the FP’s...FRET pair) separated by CaM and M13. When calcium binds, it triggers CaM/M13 interaction that increases ...same pattern described above - a sensing domain induces changes in a fluorescent signal based on ligand...biosensors to follow processes such as protein or DNA binding, enzyme or transcriptional activation, conformational...

... here. Here's what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated...Liu Z, et al. Cell. 2022.  Find JEDI-2p viral preps here (and here!)Find JEDI-2p plasmids here!  Liu,...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...EF1ɑ-driven viral vectors (AAV1) - and you can also find several other constructs in plasmid form. ...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...

... panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers are completely...a value of “1” indicating those markers are identical and completely indistinguishable. This tool is of...low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87; therefore... beads that will either bind any antibody (positive population) or won’t bind anything (negative population...panel, there are a few critical factors to keep in mind in order to get the best data possible.  Spectral...website has a convenient tool, called the similarity index, which allows the user to compare the emission spectra...designing a panel.     Figure 3: Similarity index across 12 different colors on a three laser spectral...

... Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...found yourself bamboozled by all of the different kinds of information on our plasmid pages? Well, to help... publication page, and finally on down to the individual plasmid page. The depositing lab page contains... in the collection, but you can use this name to find this plasmid from our search page (check out this...ways you can search our website). A quicker way to find this plasmid through search is... The Addgene plasmid...searching for, this is the quickest and easiest way to find it! But what are all those flames about? Flame...although you may have an entirely different use in mind! This section of our plasmid pages is relatively...

...researchers do these things with nefarious goals in mind. Indeed, it’s not always obvious that we’re doing things... sciences and featured speakers from academia, industry, nonprofits, and publishing. The livestream video...preparation for my talk and at the minisymposium. You can find a variety of additional resources and information...that someone has replicated it precisely. You can find a great discussion of different types of replication...purposes we’ll call a study reproducible if its findings can be predictably applied to future work. Importantly...expect that every study should be reproducible. Indeed, researchers are pushing the limits of knowledge...reported by drug companies attempting to apply the findings from these studies, are quite low (<30%, Prinz...

... membrane will bind to the proteins in the buffer, blocking the membrane  from binding to the antibodies...antibody binds to protein of the correct size. Many vendors will show that the antibody binds to transiently...slightly more popular due to its durability and high binding capacity. In addition, PVDF membranes can be repeatedly...unbound regions are “sticky” and have the potential to bind nonspecifically to the antibodies used for staining...But which antibody should you use? You will likely find several different antibody options targeting your...interest from a variety of vendors and you may even find plasmids encoding antibodies from Addgene that you...chains. The protein regions available for antibody binding, or epitopes, differ between a protein in its native...

...downstream of inducible promoters: the BxbI gene downstream of the anhydrotetracycline (ATc)-inducible promoter...sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies on a plate and... TP901 gene downstream of the arabinose (Ara)-inducible promoter (PBAD), and the A118 gene downstream ...downstream of the diacetylphloroglucinol (DAPG)-inducible promoter (PPhlF).  Output Plasmid: The output plasmid...register. Depending on the desired application, the inducible promoters we used to drive recombinase expression...register. RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output...promoters, and genes) were built with flanking primer binding sites (designed to have 20-30 nt length, ~60 oC...

...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating...generate compatible overhangs that allow them to bind. Ligase is used to make bonds between the insert...followed by the restriction sites EcoRI, XhoI, and HindIII. To place your gene in the proper orientation downstream...EcoRI site just 5’ of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you... backbone as well as the insert with EcoRI and HindIII and, when you mix the cut products together, the...two EcoRI digested ends will anneal and the two HindIII digested ends will anneal leaving the 5’ end of...

...high-affinity for calcium binding and developed a version of the protein with as few calcium-binding sites as possible...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the...and his colleagues began by isolating a calcium-binding protein, troponin C (TnC), from muscle cells. They...imaging for beginners Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans,...his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing...the transcription factor cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making...Metals Maarten Merkx of Technische Universiteit Eindhoven has similar interest in FRET-based sensor proteins...

... crucial recognition region is disordered, Cas9 binds DNA very weakly, most likely through unstructured...regions.   Cas9’s transition from weak, random binding to precision gene editing requires three major ...3. Though Cas9 is not a static molecule in any binding state, RNA loading stabilizes the closed conformation... the open conformation (Cofsky et al., 2022), indicating that there is more to learn about this process...helix, purple; sgRNA, dark blue.   The REC lobe binds tightly to the repeat:anti-repeat duplex of the ... stem loops and the guide region. The stem loops bind along the outside of the protein, spanning both ...Figure 3 shows the guide RNA stabilizing the DNA-binding protein portion of the CTD, ready to check for ...

...reversible and would avoid genomic off-targets or indels introduced through non-homologous end joining (...orthologs, and 7 Cas13c orthologs. They hoped to find a stably folded ortholog that cleaved RNA robustly...also increase off-target editing within the target window, possibly due to longer stretches of duplexed RNA...that editing is mediated by ADAR2DD, not PspCas13b. Find the plasmids for the RESCUE RNA editors here! Testing...duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of the tested constructs...system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note that RESCUE... RESCUE system small enough for viral delivery.  Find the RESCUE RNA editing plasmids here! Future directions...