... was the molecule of interest (analyte), and accumulation of ATP activated GFP expression (receptor), ..., then the transducer could be a microscope, plate reader, or any other instrument which measured GFP ...advantages over other mechanisms. For one, many viral platforms, such as AAV or retrovirus, can either permanently...Fluorescent Biosensors for Neurotransmission and Neuromodulation: Engineering and Applications. Frontiers in...

...of your library cargo to isolate the effects of each variable being manipulated. Thus, the adjustable nature...expression control Researchers are always wanting to manipulate gene expression – turn it up, turn it down, knock...PMC2075523. Bulcha, J.T., et al. Viral vector platforms within the gene therapy landscape. Sig Transduct...

...of interest (4) isolated your antibody:protein:DNA complexes and finally (5) isolated the DNA from your...DNA are no longer bound, and the DNA can then be isolated and studied through a variety of downstream methods...downstream analysis via any methods suitable for isolated and purified DNA, such as sequencing or qPCR. ...

... to glance at as you work. Draw your plate maps onto your plates, alongside your physical copy. If you...reminders on a sticky note   Visual Planning  Plate maps, pre-labeling tubes, and even drawing out experimental...example, setting up your pipette tips to match your plate layout can help you track samples as you add them...

... in the nucleus. The Roth lab showed that this platform accurately identified 120 known GPCR targets demonstrating..., the receptor fused to APEX2 catalyzes the biotinylation of proteins near the GPCR. These proteins are... GPCRs for chemogenetic studies to probe the relationship between neurons and specific behaviors. Chemogenetics...DREADDs are ideal for chemogenetics as they are relatively insensitive to their endogenous ligand, acetylcholine...

...multiplexing vectors are larger because they require regulatory sequences to allow for expression of multiple...example of how this works. Make sure to order 5’ phosphorylated oligos or treat with T4 PNK. Delivery options... are easier to use across multiple expression platforms: transfection, lentiviral transduction, and AAV...Multiplex gene editing with CRISPR-Cpf1 is one of the latest developments in CRISPR technologies.  It’s a simple...

...samples through our lab using barcoded tubes and plates. The critical pieces of information for a given...100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then ... database of over 2000 primers. Two 96-well PCR plates are prepared with the primer stocks for forward...shipped along with their corresponding glycerol plate on dry ice to the sequencing provider. Each sequencing...

...mechanism? Where is the control happening? Is the regulation of an exogenously introduced gene or over the...generate a frameshift mutation that will permanently ablate protein expression. If you need the ability to...different amounts of tetracycline. Tetracycline regulation is reversible (when the drug is removed expression... These control systems function at the post-translational level, unlike most of the other mechanisms described...

... with post translational modifications. One of the most common of these is 2’-O-methylation, where a methyl...once you’ve chosen a gRNA and modification(s) Congratulations! You’ve picked your perfect guide; now check...Spatiotemporally Controlled CRSIPR-Cas-based Genomic Regulation. ACS 6, 5, 695-703 (2020). https://pubs.acs.org...

... TAK 1, an enzyme that controls apoptosis and regulates transcription. Without TKA1, cells proliferate...cancer as a broad phenomenon and not only as an isolated disease. While the tumor may seem to take center...microRNA transfer: A mechanism of environmental modulation of hepatocellular cancer cell growth." Hepatology...resources on the Addgene blog Read our cancer related blog posts Learn about our cancer collection pages...

...The modularity of antibodies means that it is relatively easy to physically divide up the different regions...you to use just a fragment of the antibody in isolation from another portion. For example, Fab fragments...but not the Fc region (more on when that might be later). Find scFv plasmids at Addgene Depending on how...fragments intended for use as primary antibodies are relatively uncommon. Though, if you want to use Fab fragments...

... that underlie complex regulation in biological systems. The CombiGEM platform provides a means to create.... CombiGEM-CRISPR: The marriage of two simple platforms CRISPR has revolutionized how we decode the genome...Wong et al., 2016). With CombiGEM and CRISPR platforms now being integrated, we look forward to the realization...

... not linearly related to power, the conclusions reached at high power may not translate to real applications...widefield imaging fluorophores are excited with relatively low light levels (for a prolonged time). Other...application (Goedhart et al, 2012). The third issue relates to the environment of the fluorescent protein. ...

...where I gained invaluable experience working on and later chairing the Advocacy Committee. I’m now furthering...more. Having met many people at that conference, I later became involved with the NPA beyond the meeting....more productive and makes it easier to build relationships. Nonetheless, the committees would not function..., I had to learn how to develop new working relationships with these individuals who often had different...

...pluripotency marker expression and response to LPS stimulation, respectively. Primary cell type Gene targeted... a fluorophore or tag. To utilize HDR, a donor template containing the desired sequence flank by homology...Nanoblades which carries a gRNA, Cas9, and a HDR donor template DNA can be generated by incubating Nanoblade particles...facilitated HDR with either a ssDNA or dsDNA donor template. In vivo editing of mouse zygotes and mice with...

...pockets. They allow researchers to identify and isolate very specific biological targets. However, as popular...identical antibodies (See Fig 1.). rAbs are a relatively new technology that takes advantage of DNA sequencing... NOT align with the atlases/literature and is unrelated to the target protein, we categorize the antibody...strong signal. However, exposure time and its relationship to signal depend on what you’re comparing your...

...acquired divergent functions due to mutations accumulated over time? Undergraduates can help to answer ...If the phenotype is corrected, students can be relatively confident that the gene from the test species...the S. cerevisiae met deletion strains and then isolate transformed strains on media that lack uracil, ... MET genes to grow.) Students then use replica plating to determine if expression of MET/Met genes from...

...genetic tool for researchers to conditionally manipulate gene expression in vivo using site-specific recombination...orientation, the SSR will excise the DNA fragment. By manipulating the number, orientation, and type of target ...morphology, and circuit mapping of a targeted neuronal population.    Figure 2 (modified from Schnutgen...study the physiology or behavior of certain cell populations.  Acknowledgement:Through a partnership with...

...double-stranded break. HDR copies the sequence from a repair template with flanking sequence homology for error-free...systems, and NHEJ will be favored even if a repair template is present. HDR also presents a cloning dilemma...CRISPR gene editing. In organisms where HDR is downregulated, MMEJ represents another method for making targeted...’s become clear that the power of this editing platform lies in the diversity of nucleases and their applications...

...glycosyltransferase genes and other proteins related to cellular glycosylation. Get the library at Addgene CasRx...requires the much less efficient double stranded templates, rather than the more efficient single-stranded...cellular protein. They used CRISPR with a ssODN template to introduce the small wrmScarlet11 which is less...