...created a yeast-based directed evolution approach with some notable improvements. Directed evolution techniques... fluorescent protein--no affinity purification required! The team used other molecular techniques to introduce...microtubuli and plasmids tagged with MapTau to label entire microtubules. Find these fluorescent mammalian ...contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag...

...). These so called “plasmid addiction systems” require dividing cells to retain plasmids - if a cell does...Figure 2). This MazF-ZeoR cassette is flanked by two direct repeats which can be used for marker removal so..., knock in a GFP cassette, and introduced site-directed mutations, all without introducing unwanted selection...short-lived RNA antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation...Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of...

...incredible versatility opens aptamers for use in many entirely new applications including aptamer based fluorescent...sensitive detection assay. In 1999, Grate and Wilson paired laser cleavage and an RNA aptamer that bound the...to induce fluorescence outside the context of a paired fluorophore protein. Years later, Samie Jaffrey...mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein equivalent... less immunogenic than proteins. Assays that require recognition of ions or particularly small molecules...

...bacterium, and is essentially always there, or acquired, which means the bacterium is not naturally resistant...intrinsically resistant to this class of antibiotic. Acquired resistance can be the result of one bacterium ... died for instance). These three mechanisms of acquired drug resistance are referred to as methods of ... to a drug while a full course of treatment is required to wipe out those bacteria that are partially ...Kirby-Bauer susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results. While ...

...contamination: Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance and Metastatic...from the official biorepositories or have been directly isolated from patients. In these cases, a test...outlined in this article. Now many journals strictly require evidence of cell line provenance and identity. ...Council (BBSRC) and Medical Research Council (MRC) require authentic cells to be used and STR profiling to...can be prevented and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach...

...Historically, molecular interactions were detected by indirect means often using probes with the potential to...proteins, pioneers of this technique were able to directly detect protein/protein interactions in living ... to each other in the spectrum, you can easily directly excite the acceptor with the laser used to excite...corrective approaches and appropriate controls are required to make this method useful for dynamic experiments...fluorescence, this method is also less sensitive to direct acceptor excitation artifacts and it is possible...

...sample would directly correlate to protein abundance on your membrane, which would be directly reported by...housekeeping proteins.” Fully quantifying a western blot requires a standard curve with a pure sample of your protein...proteins may not transfer precisely evenly across the entire blot. So how can you accurately compare different...your approach) as your final blot. Quantifying the entire lane of protein provides a loading control that...

...interest (Fire et al., 1998). The discovery of RNAi in the worm actually rewarded depositors Andrew Fire and...Yeast are also the first eukaryotic genome to be entirely sequenced and is very amenable to genetic manipulation...utilized the worm to study calcium signaling in the entire adult brain, using a plasmid that expresses a genetically...(1974): 71-94. PubMed Central PMCID: PMC1213120. Fire, Andrew, et al. "Potent and specific genetic interference...

..., BioID2 exhibits enhanced proximal labelling, requires less biotin, and allows scientists to more selectively...stability. Thus they conducted multiple rounds of directed evolution until they identified a mutant with ...APEX2, although it can rapidly label proteins, requires H2O2 which can be toxic to living cells. To circumvent...issues, the Ting lab utilized yeast display-based directed evolution to generate two mutants, the 35 kD TurboID... PMCID: PMC4831873. Lam, Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and...

...a stupendous listener, but I aspire to  be. If you don’t at least aspire to be a good listener and consciously...each other disagree about a specific decision, direction, or goal. Resolving these types of conflicts is...Assumptions: Making incorrect assumptions can be a direct cause or consequence of lapsed communication. Making...job”. It’s certainly resolvable, but will likely require a manager to explicitly assign the responsibility...

...3 days to complete the entire experiment, but you’ll have to spend the entirety of each day doing the ...work in a job where you’re pulled in a thousand directions at the start of the day, writing your to-do list...depositors from Boston University Contact Dr. Gilmore directly to see if he knows anyone who’s interested in ...if I had planned my day extensively, my lack of desire to go sit in the tissue culture hood may have prevented...

...instability at 37°C (Ouellet et al., 2016) and requires a tRNA scaffold to improve its stability in vivo...Ming Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach. In doing so, they identified...green fluorescent aptamer, was identified through directed evolution. First, a pool of RNAs that activate...Fluorescence is observed when these RNAs complex and the entire aptamer can hybridize and fold. (Alam et al., 2017...fluorescent protein by fluorescence-based selection and directed evolution." Journal of the American Chemical Society...

...Vitamin B7) is first an essential coenzyme that is required by all organisms for life. Biotin is only synthesized...Most organisms have less than five enzymes in the entire proteome that are biotinylated. In E.coli for example...responsible for biotinylating only one lysine in the entire organism on the biotin carboxyl carrier protein...recall, BirA biotinylates only one lysine in the entire E.coli proteome. Using this knowledge, scientists...scientists identified the 13 amino acid sequence required for BirA recognition and biotinylation. This 13 amino...

...system (OTS) for the incorporation of phosphoserine directly into recombinant proteins (5). Optimization of...of this system defined three components required for translation (Figure 1): A tRNA that recognizes a... The generation of pSer-incorporated proteins requires a molecular toolkit containing the OTS, an appropriate...phosphoserine OTS, Sep-OTS, contains the components required for the translation of pSer-incorporated proteins...phosphoserine must be recoded to ‘TGA’ using site-directed mutagenesis. In the C321.ΔA strain, this TGA will...

...studies, where it could potentially label cells not directly connected to the injection site, or when using...volume. You’ll often hear that “high titer” is required for efficient AAV transduction, usually without...the optimal number of viral particles per volume required to express the gene of interest, in the cells ...are a few factors to consider. First, the titer required is inversely related to transduction efficacy:... recommended to optimize to the “lowest number required” instead of using the first amount that looks ...

...transformants for single colonies (this usually requires 10-20 plates) (see box below). This initial transformation...growth, scrape up, pool and extract plasmid DNA directly from the colonies. Transform the resultant plasmid...a lysis buffer to release recombinant proteins directly onto the nitrocellulose for subsequent probing... can lead to high background that obscures the desired signal. We have successfully corrected this by ...organism, it can be helpful to spot this protein directly onto nitrocellulose and carry out the steps from...

...way to verify that the insert ended up in the direction you want - usually by Sanger sequencing or further...course there’s much more detail and verification required for the process to work well, so let’s go over... that the digest go until completion. The time required for complete digestion varies for different enzymes...associated manufacturers websites. These stains require you to either stain your gel after you run your...above, respectively). Some of the above stains require you to visualize your DNA using UV light – please...

...membrane, a threshold is reached and the neuron fires, sending the electrical signal down the axon (Lodish...subsequently photo-stimulated through fiberoptic cannulas directly implanted in the injection site and connected ... et al., 2008; Taylor et al., 2016; Hooper and Maguire, 2016). Recombinant adeno-associated viruses (rAAVs...Central PMCID: PMC4971574. Hooper, Andrew, and Jamie Maguire. "Characterization of a novel subtype of hippocampal...PubMed PMID: 4940442. Schnütgen, Frank, et al. "A directional strategy for monitoring Cre-mediated recombination...

...Transposition protein: This is the protein that’s required for the transposon sequence to move from one DNA... transcriptase and integrase enzymes which are required for LTR retrotransposon transposition. Non-LTR...animals Transgenic animals are often generated by directly injecting DNA into the pronuclei of fertilized...dystrophin, the gene mutated in muscular dystrophy, require truncation to fit in viral vectors, but easily ... into eukaryotic and prokaryotic genomes. They require just two key components, the transposon’s DNA sequence...

... two very similar proteins. Additionally, they require variable expression of your target across samples...and it causes a large deletion that removes the entire exon that codes for the epitope recognized by your...expected target protein in a Western blot. You acquire a plasmid encoding your protein of interest, known...you very similar results, while the third looks entirely different (B), giving you confidence that your...amenable to high-throughput analyses, however, it requires access to a mass spec facility and the ability...

... Material Transfer Agreement process that kits require; Nicole, the web master in charge of all our externally...You might find that your next paper is going to require help from a biochemist, a geneticist, a microscopist...world, so a process or project or mission that requires a broad knowledge base is going to necessarily...more things necessary to make a successful team. Entire books have been written about this! But this is...the type of team. Knowing what roles your team requires to function is certainly necessary, but equally...

...concentration of your reagent. Be aware that this approach requires excellent attention to detail, since you’ll end...Transfers are a little trickier than gels and may require multiple runs to optimize or troubleshoot.   Before...migrate…so don’t try eliminating one component entirely. If you are trying to transfer proteins of vastly...antibody.  Pro tip! If your antibodies are coming directly from tissue or supernatant, and you’re doing the... you don’t need to compare the protein amounts directly, stripping and reprobing. Just make sure to go...optimizing it. Blots for quantification generally require more optimization than blots used to confirm overexpression...

... a variety of Cas enzymes with different PAM requirements and editing abilities, such as base editing,...with multiplexed guides, mini screening libraries, direct edit optimization, and more. While there are many...modular nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system...fantastic way to save time and money on studies that require many assemblies or that compare the effects of ...construct. Once you’ve identified your experimental requirements, it’s a good idea to select an available kit...

...in a hyper-competitive environment where getting hired hinges on performing near miracles. When evidence...defending the role of science in our democracies will require constant vigilance and actions beyond marches, ...idea, but they might feel disconnected from the entire scientific process.  I've spent a lot of time debating...debating how to fix this.  As scientists, we work tirelessly in the lab where the groundbreaking research ...training as an academic scientist. I’ve worked tirelessly to get the publications, grant funding, and teaching...

... to optimize and troubleshoot the system. Site Directed Mutagenesis by PCR & Single Base Editing with ...Interestingly, this technology may make some uses of Site Directed Mutagenesis by PCR obsolete! Honorable mentions...give a great science talk from Addgene Executive Director Joanne Kamens. I highly recommend taking a look...August posts in chronological order here: Site Directed Mutagenesis by PCR Google Forums Round Up: First...

...the success of the company is attributed to the entire Addgene family; the depositors and requesting scientists..., Addgene supporters, and of course, Executive Director, Joanne Kamens, and founders, Melina Fan, Ken ...headed to the Museum of Science in Boston, where the entire Blue Wing was set aside for the event. As guests...family Perhaps the most heartfelt portion of the entire night was when Melina and Ken called their parents...

...community, Blugene will regularly be paired with a variety of science-inspired friends. As you can see on the... also made it hard for users to get where they desired. It became clear to us that we needed to improve... you can find what you need and feel happy and inspired to tackle your next science challenge. If you ...

...everyone involved. We’re adding self service to the repertoire of tools you can use to get support for our resources...our various FAQ categories or search through the entire FAQ collection. You’ll find topics ranging from...and press send. Doing so will bring your concern directly to our support team’s attention, where we can ...can then offer the same high level of direct service you’ve come to expect from us.   Try out the Help Center...

...resulting from past practices and mores.  His list is dire:  The current system is unsustainable, hypercompetition... tackle the issues with rules and practices of indirect costs.  The peer review and grant awards systems... encouragement.  Dr. Sibby Anderson-Thompkins, Director of the Office of Postdoctoral Affairs at UNC, ... put on good mentoring and applauded the new requirement for PIs to include in their annual NIH progress...

...disease” often requires understanding the disease genetics and the “treatment” usually requires drugs. While...validated for specificity to their target, or their direct target can be identified if it isn’t known. If ...undruggable’ proteins – having multiple hits or an entire pathway of possibilities greatly increases the ...