...recombinase polymerase amplification (RPA) using a commercially available amplification kit. In this case,... RT-qPCR tests to diagnose COVID-19 and some serological tests to determine past exposure. However, the... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...reverse transcriptase to produce cDNA. Isothermal amplification is done at one constant temperature, eliminating...rapid detection.  Highlights: Cas13a is not catalytically active in the presence of two or more mismatches...that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA binds to... SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low as 1.2 copies DNA...

...selection and directed evolution." Journal of the American Chemical Society 136.46 (2014): 16299-16308. PubMed...di-GMP and cyclic AMP-GMP." Journal of the American Chemical Society 135.13 (2013): 4906-4909. PubMed PMID...aptamer soup by exploring the development and applications of fluorescent RNA aptamers in Addgene’s collection...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...biotin (the addition of biotin allows for the purification of specific RNAs from cells using this tag, ...TO1-biotin and TO3-biotin contain an acetate modification that reduces nonspecific binding with RNA by...non-coding RNAs in mammalian cells." Nature communications 9.1 (2018): 656. PubMed PMID: 29440634. PubMed...

...established applications of the original hybridoma monoclonals, we anticipate that our chemically identical...kidney development. Despite their ubiquity and significance, these receptors have proved challenging to ...specific ligand-binding functions, all key to basic biomedical research and drug development. Integrin structure...long legs that protrude from the cell membrane, physically connecting the extracellular environment and ...signal cascades, and allowing agile responses to mechanical forces in the cell’s microenvironment. The actin...and (C) extended-open. When a ligand binds, it typically shifts the integrin from its closed to its open...resulting in cells that elongated and morphed asymmetrically (Jo et al., 2022). By contrast, LVDP-binding...

..., the lessons learned in pursuing one technological application can benefit many others and accelerate...vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs HIV-1 infects...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...create mutations in genes essential to HIV-1 replication. However, this approach could permit some viral...along with Cas9 in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed...-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, although the ...these methods represent exciting advances in preclinical HIV-1 research that should be pursued in animal...

...simulate physiological gene expression regimes that cannot be easily achieved with classical gene overexpression...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 ...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...some reporters are better suited for particular applications. Read on to learn more about each reporter or...labeled tyramide molecule into a highly reactive radical, which is then covalently linked to residues within...however, compatible with most multi-labeling immunohistochemical protocols where either a directly labeled...

...would need to be much, much lower before clinical applications could be considered. It is important to ...answer questions of efficiency and applicability. Given moral and ethical concerns surrounding such research...experiments are needed before we can consider clinical applications for germline editing. Although this paper...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were... embryos carried indels at the mutation site, indicating that repair proceeded through NHEJ rather than...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure...consider that already-tested CRISPR alternatives are applicable in many situations. In the case of hypertrophic...

...previous two posts in this series described a practical approach to selecting a bright fluorescent protein...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity and has...most likely in an antiparallel configuration), typical structures known as Organized Smooth Endoplasmic...Constantini et al, 2012). The localization and biological properties of new fusions can be compared with...

...the 4D nucleome. Visualizing specific loci has historically been difficult, as techniques like fluorescent... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing... The technique successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated...distances correlating with the previously established physical map. In comparing pairs of targets ~2 Mbp apart...the respective hairpin binding domain. Pairs of identical hairpin sequences specify the primary colors blue... colors and a white composite. gRNAs with two identical hairpin binding domains produce blue, green, or...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live...

...in their ability to be specifically and potently activated by pharmacologically selective effector molecules...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...wiring of the brain and the underpinnings of neurological disease, neuroscientists need to be able to ...What is chemogenetics? Chemogenetics relies on genetically introducing engineered receptors that are only...et al., 2019). Using PSAMs PSAMs PSAMs, or pharmacologically selective actuator modules, were developed... such as varenicline, are already approved for clinical use. Varenicline has been further modified to ... create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download our chemogenetics...

...is complex Plasmid classification has shifted over the years, and new classifications are still being created... Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4)...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...genes between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred...which strains of E. coli contain this F plasmid is critical for successful Gateway cloning. In addition, scientists...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...there are two main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...excitation or inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression...recombinase on transgenic flox animals to introduce modifications at the DNA level in a temporally and locally... brain area, avoiding side effects from such modification in other areas. An alternative strategy is to...in a certain type of behavior (for a review see Stoica et al., 2013). Vector delivery may also be achieved...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...remove the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et ...PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh...

...one be interested in state machine technology? Biological research has produced a massive amount of information...processes that are influenced by temporally ordered biochemical events. How do RSMs work? The state of an RSM...prescribed region of DNA termed the “register”. Each chemical input to an RSM drives expression of a distinct...performed on one register at a time. If a particular application makes it difficult to isolate single registers...state read-out method may not be suitable for applications that require minimal cellular perturbation. ...The input plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream...contains the register. Depending on the desired application, the inducible promoters we used to drive recombinase...

...relationships as part of the job application process can dramatically increase the chances of being seriously...demonstrate in your application). Beware the myths of scientist “overqualification” but it may not be ... of a resume can be crucial for a successful application. It takes time to prepare an excellent resume... Many job postings will result in hundreds of applicants the day they are posted.  If your resume is not... here for more on timely submission of a job application. 3) Read job descriptions carefully Be open minded...bold. You don’t have to have all of the listed qualifications to apply for a position.  A job description...wish list and the hiring manager knows that no applicant is likely to meet all the criteria. Note the criteria...

...must make is which system you will use, or more specifically, the type of cells that will be used to make...spanning both prokaryotic and eukaryotic organisms. Typically, what system is used is dictated by the specific...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do... one passage (transfection). This differs from typical insect cell preparations, which we talk about below... below. Processing The viral supernatant is typically collected over the course of a couple days to increase...supernatant will depend on the virus type. AAV is typically purified using methods like ultracentrifugation...

...Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. Stem...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...differentiate into cells of peripheral blood. Typically, there is a very small number of ASCs in each ...Limited. Only specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC ...of post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient...bypass the transplantation compatibility issues and ethical concerns of ESCs. As iPSCs can be sourced directly...

... many biological research situations, the relevant volume is that of the cell. For the physical behaviors...experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, ...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics...monomeric FPs are not actually monomeric, at least in practical terms. The propensity of a particular type of ... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...structures are large (microns), bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to...

...strengthened my critical thinking and problem solving abilities, increased my technical know-how, and helped...the bench, look for “technical or product support specialist,” “field application scientist,” or “scientific...scientific or technical writer,” among others. Don’t discount job postings looking for PhD applicants since many... helped develop my science communication skills. Ultimately, my degree prepared me to be exactly the type... many will also accept applications from master’s candidates with experience.  Use LinkedIn to see what... you’re interested in (i.e. biotechnology, pharmaceuticals, etc.) Do their jobs sound interesting? Is ...deeper foundation of scientific knowledge and technical know-how that increased my autonomy and satisfaction...

...outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-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...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts ...