...production of dsRed E5. This timer predictably transitions from green to red fluorescence (500 to 580 nm...elegans embryos. However, this initial timer was a tetramer with a propensity to aggregate in cells (Tsuboi... expression time but independent of protein concentration. Calibration curves correlating this ratio to...have realized how problematic aggregation-prone tetrameric fluorescent proteins can be. Cytotoxicity, improper...to red (see Table 1 for emission and absorption spectra). Subach et al’s directed mutagenesis also produced...Miyatsuka et al.) Distinguish between models for trafficking to the lysosome by fusing Medium-FT to LAMP-2A...structure and a particular biological event (trafficking to a subcellular location, start of gene expression...

...the non-profit sector are transferrable to industry such as technology transfer, communications, medical.../experience, i.e. a research role, for a Ph.D. trained scientist. If the candidate is ultimately looking... on projects with an international scope (i.e. strategy/management consulting firms), sponsor more H-1Bs...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an...management, collaboration management, grant administration, clinical operations, and so on. Candidates...patents and letters of recommendation. EB-1A extraordinary ability category The EB-1A - is similar to national...expertise in field). Publicity in major media and/or trade journals: You the individual must be named in the...

...ligand (after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion...Vanderbilt Eye Institute and Tonia Rex's lab. A common strategy to determine the binding kinetics of a purified... avidin (or its derivatives streptavidin and neutravidin) is for all practical purposes covalent. Therefore...so, theoretically, saturated avidin could form tetramers of biotinylated protein. However, oligomerization...for improved biotinylation selectivity A common strategy to biotinylate proteins involves amine-reactive...subsequently purify the fusion protein from a bacterial extract. The MBP tag can be removed using Xa protease. ...joyful that getting to the final step and the transformed bacteria refuse to grow. Eventual success was...

...reverse transcribe their gRNA into DNA, which is then transcribed into mRNA before being translated into ...genome types that can be either double stranded (ds) or single stranded (ss) (ie, dsDNA, ssDNA, dsRNA, positive... of virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case...looking for molecular biology tools, a very useful trait. Virus structure While all viruses have a genome...2016 Viruses with DNA genomes can either follow central dogma (gDNA to RNA to proteins) or require an intermediary...generally still kill the infected cell.) This is in contrast to bacteria and eukaryotes, which use a binary...and envelope), has a “gutted” genome with genes central to the replication process removed. This means ...

...amplification) or reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified... specificity to detect nucleic acids at low concentrations and/or are too expensive, time-consuming, and... method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). Both SHERLOCK...mutation of interest. Together SHERLOCK and DETECTR demonstrate the power of CRISPR-based diagnostics. SHERLOCK...almost anywhere as lateral flow strips are easy to transport and work rapidly, providing reliable results in... the SHERLOCK detection system The Zhang lab demonstrated that SHERLOCK could reliably distinguish between...    DETECTR- DNA Endonuclease Targeted CRISPR Trans Reporter Cas12a (Cpf1) is a CRISPR Cas variant that...

...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...organismal systems described at this meeting were extraordinarily diverse. Plans to develop tools to study their...) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes...Poison frogs are great for studying parenting strategies and make important ecological indicators since...(which is considered recent). They have diverse traits with good genetic mapping morphology and interesting...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper snail...

...discovery, Addgene and RSRT have consolidated and centralized a collection of molecular biology tools so that...maturation (Tillotson & Bird, 2019) and is a global transcriptional regulator of thousands of genes. Studies have...have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...groove of AT-rich DNA sequences to regulate transcription (Baker et al., 2013). Mutations that affect .... Much research is now focused on therapeutic strategies that use gene replacement therapy, gene editing...editing, MECP2 reactivation, RNA editing, RNA trans-splicing, and protein replacement to reverse the impacts...in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy. In 2014...

...than the signal generated by the energy transfer. On the contrary, if you choose a pair that are too far...Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer    Interesting Articles...FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules...distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular fluorophore...absorbed light, at a given wavelength, to the concentration of fluorophore in solution) are two additional...fluorophores with significant overlap in their spectra. The pair CFP-YFP was the first to be used to study...cross-talk but you also don’t have any resonance transfer. You have to find a balance between FRET efficiency...

...subtle difference in mouse chow, a different water filtration system, environmental stability in an incubator...is not just a question of being honest and straightforward, it’s an important part of being a “good scientific...differently. Communication comes in many forms—from transparency about data to allow others to see what you actually...communication and sharing fits together to make for a transparent and collaborative research environment. Journals...Currently, most of the burden for pushing for more transparency in science communication falls on the shoulders...developed STAR Methods to improve the structure and transparency of methods sections, including clear information...researchers are not strongly rewarded for being transparent in their communication or making their data and...

...Technologist/Designer     Fieldwork and traveling Many people are attracted to science because they enjoy spending...job involves traveling, showing that you are a highly organised and experienced traveler by citing specific...skills include: Science Reporter Scientific Illustrator Research and Development Scientific Advisor...Experience in the field or in a position that requires travel can show that you are self organised, reliable,..., including: Livestock Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist...writing skills Writing a thesis or dissertation demonstrates that you have advanced writing and formatting...job title actually involves, other than what is portrayed in the media and in films, which can often be ...

...target protein expression levels, assessing post-translational modifications, determining cell health, analyzing...a pressurized buffer called sheath fluid and transported through tubes or capillaries to a laser. As the...continues in the same direction as it was initially traveling is called forward scatter (FSC) and provides information...scatters sideways to the path that it was initially traveling is called side scatter (SSC) and provides information...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...antibody thus amplifying the signal.       Intracellular protein targets, measured by fixing and permeabilizing...permeabilizing membranes For intracellular protein targets, users must first fix proteins within the cells...

...injections can be found here [5, 6]. Opsin transgenic mice Transgenic mouse lines have also been engineered...vectors for anterograde axonal tracing with fluorescent proteins in nontransgenic and cre driver mice. Current...and versatility, the technique is remarkably straightforward to learn and relatively cheap (compared to ...Director of the National Institutes of Drug Abuse Intramural Research Program (NIH-NIDA IRP), with the stated...specific brain region can result in a particular neurotransmitter release or post-synaptic current. Fortunately...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector...to express. For example, if you inject into the ventral tegmental area (VTA) but wish to stimulate VTA ...

...challenging to stain intracellular compartments in in vivo model systems, so extra care must be taken to...target through binding of the molecular species. Administration of these dyes results in direct binding between...variant tag that covalently reacts with a small substrate ligand motif attached to a fluorophore. When paired...advantages to using these self-labeling proteins over traditional fluorescent protein tags, such as higher specificity...on a protein, enabling the labeling of protein substrates/peptides and antibodies. Example applications...green, red, and far-red dyes is important for intracellular fluorescence live imaging ​​(Hayashi-Takanaka...fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like Rhodamine 123 (R123) (Liu et al...

...Cancer Colleciton Pages Use the ClonTracer Library for Lineage Tracing Learn About Cancer Immunotherapies... with vulnerabilities) to design a therapeutic strategy that works. But, those darn cancers have revealed...patients still don’t benefit from this approach. One central bottleneck is that most recurrently mutated cancer...are likely gain-of-function, loss-of-function or neutral, as compared to wild type, would be extremely helpful...the cancer plasmid collections listed above to demonstrate how gene expression profiling, systematic tumorigenesis...morphological profiling can be used as generalizable strategies to map the function of rare cancer variants and...MH, Elife, 2017. PubMed PMID: 28315521. PubMed Central PMCID: PMC5386591.  The genetic pathway reference...

...for mutation validation, we found these assays frustrating to work with, time consuming, and minimally informative...performed to amplify the target using PCR. Instead of tracking the reaction progress as in real time PCR, each...edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for “functional... an unedited site distal tothe predicted double strand break, and a NHEJ/ “drop-off” probe designed to...to bind directly at the predicted double strand break site (Figure 2A). The single molecule resolution... 291–293 (2014). PubMed PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, ...PubMed PMID: 26914317. 4. Mock, U. et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates...

...cleaves ssDNA in trans. In sum, CasPEDIA documents the target substrate (DNA/RNA and strandedness) along with...Cas enzymes) cleaves both strands of its dsDNA target in cis and has no trans activity. AsCas12a, on the...Cas enzyme: Nuclease properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability...features: CasID CasPEDIA will display both the cis and trans activity of each entry. For example, SpyCas9a (one...with its action on the substrate (nick, bind, cleave, mixed). This information represents the ‘big picture...required? CasPEDIA provides information on crRNA and tracrRNA requirements, along with any other required non-CRISPR-associated...common applications of an enzyme for your non-traditional experiment. Perhaps you are a structure junky...

...cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:ATP ratio. Most of... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...sensors developed for it. Calcium triggers muscle contraction, and calcium channels are activated for neuronal...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...

... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses...Aprataxin, Tdp2) that function to make ends better substrates for ligation. Although we do not describe these...introduced. Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated... the deletion can be less heterogeneous when constrained by sequence identities in flanking sequence (... “double nickase” strategy vastly reduces breaks and mutations at off-target sites. Christina Mork contributed....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...

... look like (the 3rd sequencing trace). While it requires a little extra wet lab work, this is still a ...your edit of interest. For all of the screening strategies outlined below, the technique can be used as ...do. A popular screening method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et...et al., NAR). This technique uses the trace file generated in Sanger sequencing and a simple algorithm...Fig. 1: Overview of TIDE. The input is sequencing trace files of WT and edited cell lines. Then decomposition...and your Cas9-targeted cells. Simply upload both trace files to the TIDE tool online along with your sgRNA...design phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to ...

...quantity of your POI, but simply whether its concentration changes in response to experimental parameters...errors like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type ...type of positive control necessary to demonstrate a POI’s absence. It also helps confirm that lanes are loaded.... No pipetter is perfect, and proteins may not transfer precisely evenly across the entire blot. So how... Figure 3: In a perfect world, protein concentration in your sample would directly correlate to protein... in the loading control are due to loading and transfer errors — not to any factors from before the proteins...you’ll need to conduct a control experiment to demonstrate that expression of your reference protein does...