We narrowed to 888 results for: URE

...rough structure of the plasmid based on the predicted sizes and organization of different features within...and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer’s manual...their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases...loading them. The glycerol in the buffer will make sure your sample settles in the gel well and the dyes...expected band sizes. Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA...approximately 75-80% of the way down the gel will ensure you keep smaller bands from running off; however...

...plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors...Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these...foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered...stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA ... used in conjunction with positive selection to ensure your gene gets targeted to a specific location ...293HEK, Jurkat T cells Inhibits protein synthesis; premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa...is often used as a supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate...

...the amino acid change based on maps of protein structure. Sometimes this provides a great deal of clarity...bioassays could accurately test for molecular signatures of variant function and cancer phenotypes. Broad...systematically as possible. In developing these assays, we figured that they would need to be generalizable and capable...capable of measuring a variety of molecular signatures or cancer phenotypes. These assays were designed...look-up-tables” of mutant function that could enable future clinicians to improve real time decisions when ...proof-of-concept. If such approaches are proven to improve future clinical decisions, we can work together to realize...of precision functional genomics and propel the future of cancer precision medicine for the next-generation...

... this class measures either factors as proxies for a cellular process or directly measure a cellular process...to the structure of actinin in low vs. high tension. These systems are powerful tools to measure real-time...boiled down pretty simply – they are systems that measure biological phenomena by generating an output which...microscope, plate reader, or any other instrument which measured GFP levels and converted that signal into a readable...will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost all...tissue preps, in multiple species, and in cell culture models. Some of them have even been expanded for...

...passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Richter MF, Zhao KT, Eton...for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at the Cell Symposia:...from the infected to a fresh uninfected host cell culture). This dilution step represents the major difference...dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038/s41587-020...

...-peptide (represented as an orange wedge in the figure on the left) and inserted it into a plasmid, creating...note that IPTG is also included in the medium to ensure transcription of the lac operon.   Tips for blue-white...care in making your plates: X-gal is light and temperature sensitive and needs to be added to media after...autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient drying...analysis, like PCR or restriction digest. Make sure you use a proper E. coli strain (i.e. contains the...STBL4, JM110, and Top10 are a few examples. Make sure you use a proper plasmid (i.e. contains the α-complementation...selection methods. Combining selection and screening ensures that the white colonies you see are white due to...

...journal. Be sure to check the Author Guidelines of the journal you are submitting to and make sure your manuscript...lab resources, such as catalogue numbers and manufacturers’ details for antibodies, plasmids (like your...F1000Research, also ask for the raw data behind the figures and results. This is a relatively new development... of science publications. Peer reviewing To make sure your manuscript is perceived as a reliable source...when publishing a scientific manuscript Dos: Make sure your paper fits the scope of the journal you are...chances of getting your manuscripts published in the future. Disregard reviewer’s comments without explaining...Always check the journals Author Guidelines to make sure that your paper fits with the journal’s house style...

...antigens.      Figure 1: IgG antibody structure with important features labeled. V = Variable ...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by...Fragment Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...tool box. As you consider which tools to use, be sure to think about what pieces of the antibody you do...Bates A, Power CA (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody ...

...Conclusions & implications One striking feature of this paper is the failure of the embryos to undergo editing...mutation in human embryos." Nature. Epub 02 August 2017. doi:10.1038/nature23305. PubMed PMID: 28783728. ...coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents...but instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies... proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes...broader applicability of CRISPR germline editing. Nature News mentions that the MYBPC3 gRNA used in the ...

...48 hours exposure to blue light also induced tail swelling in ~75% of worms (see below figure). Zhou et... current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible...is a serine/threonine kinase with a similar 3D structure to MEK1, but Raf1 and CDK5 have less similarity... pathways Negative feedback loops are a common feature of signal transduction pathways, but are often ...used a photoswitchable CDK5 in C. elegans. See figures 4B and C in Zhou et al for details on these experiments...no. 6327 (2017): 836-42. PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot...Carrasco, and Markus W. Covert. "High-Sensitivity Measurements of Multiple Kinase Activities in Live Single...

...guest blogger, Gary McDowell, executive director of Future of Research. On December 1st 2016, many postdocs...some cases reversing these decisions entirely. At Future of Research, we had been collecting data and documenting...published in F1000Research on November 2016. Due to the nature of the postdoc position, we viewed salary raising...FLSA updates was granted nationwide. Again, we at Future of Research believe that, while the mandate has... out about what their institution is doing from Future of Research over social media, not from their institution...actually getting paid, which we will be analyzing for future updates. But suffice it to say, salaries are showing...McDowell is currently the Executive Director of Future of Research. He is interested in advocating for...

...available, including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC...the CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the...by NGS before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial...and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180. PubMed...cells with a lentiviral CRISPR-guide RNA library." Nature biotechnology 32.3 (2014): 267-273. PubMed PMID...activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed Central PMCID...

...publishing, but still made it so she could “get exposure to the drug discovery process.” The ClonTracer...xenografts after cell implantation. Their goal was to figure out how to optimize protocols for in vivo screening...panned out quickly,” and was summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers...specifc, easily read sequences that can be used to measure their clonal abundance before and after a treatment... populations with resistance to both drugs. The future of ClonTracer Since starting her new role as an...populations are treated as they arise. In their Nature Medicine paper, Bhang and coworkers also looked...cancer therapy using high-complexity barcoding." Nature medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130...

...workshops (see the Careers essays in the journals Nature and Science, for example). This blog post is about...write a manuscript. Consider questions like: What figures are missing? For example, are additional controls...been validated? How will the results fit into a future manuscript? You could easily spend a day or a ...experiment. However, by taking this time, you can help ensure that you understand the methodology and can have...the data and any recent relevant studies in the literature. Plus, presentations benefit from practice. If...and how your research fits in with the current literature. Finally, if you give your talk or poster to ...application and manuscript reviews, etc. You'll also meet future collaborators and get access to unpublished information...

...particles produced could not replicate. However, Mother Nature found a way to re-create a functional virus. Indeed...non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived vectors are capable of transducing...your gene of interest into and is an important feature for your experiment; it must be chosen carefully...expression of your gene. The addition of Dox in the culture will then repress your gene of interest. This system...state and is expressed when Dox is added to the culture. This system is now used as it shows rapid gene...biosafety guidelines for more information and always be sure to consult your institution’s Biosafety Committee...cells start releasing viral particles into the culture supernatant and you can generally harvest the supernatant...

...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...retrotransposons are like retroviruses both in their structure and mechanism of replication. They contain two...and reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons... TEs have LTRs on both of their ends.    Figure 2: Overview of DNA transposon transposition. Modified...Transposon mutagenesis screens Transposons, by their nature, are mutagenic elements, which makes them a great...of DNA up to 10 kB in size in bacteria.   Figure 3: INTEGRATE system for RNA guided transposon insertion...Rev Genet 41:331–368. https://doi.org/10.1146/annurev.genet.40.110405.090448 Kumar A (2020) Jump around:...

...  Figure 1: (Top) Polyclonal antibodies are produced in animals and consist of a mixture of antibodies...made from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain...Reproducibility crisis: Blame it on the antibodies. Nature 521:274–276 . https://doi.org/10.1038/521274a Begley...Raise standards for preclinical cancer research. Nature 483:531–533 . https://doi.org/10.1038/483531a Bradbury...Reproducibility: Standardize antibodies used in research. Nature 518:27–29 . https://doi.org/10.1038/518027a Bradbury... (2015) Antibodies: validate recombinants once. Nature 520:295–295 . https://doi.org/10.1038/520295b Bradbury...

...mCherry coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site...switch conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...control expression of an optogenetic reporter would ensure that the reporter remains silent until a cell or...tissue-specific Cre is provided. Since the FLEx switch ensures Cre-dependent reporter expression, the potential...

...poly-uracil tract as shown in the figure to the right. The tertiary structure of the hairpin-DNA complex is...the transcript.   Figure 1: A predicted conserved secondary structure and sequence conserved Rho-independent...enzymes that cut RNA not protected by secondary structure. It is thought that poly(A)s are used to control...relies on a specific sequence and RNA secondary structure to induce transcript cleavage, similar to the ...motif and a downstream GU-rich region (shown in the figure below) releases the mRNA from the polymerase and...export and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal...

...field. Nobel Laureates and the ever expanding importance of RNA The second keynote lecture was presented...project on its 25th anniversary, how the NIH sees the future for genomics, and President Obama’s Precision Medicine...presented by Nobel Laureate Jack Szostak. Dr. Szostak’s lab continues its work on the origin of cellular life... two of the conference was kicked off by Nobel Laureate (and Addgene depositor) Craig Mello. Taking his...problem of antibiotic resistant bacteria. See the picture to the left for his list of the bacterial species...engineering. The final keynote presentation was by Nobel Laureate Thomas Steitz of Yale. He gave an update on new...conformation “not like DNA which has a boring structure.” That certainly summed up this conference—nucleic...

...little better. As a Master’s student, she had less pressure to publish and more freedom to attend conferences...legitimate brain exercise in developing a plan for the future. From good ideas to a SynBio startup At some point...them to see how feasible they were, conducting literature reviews and having weekly journal clubs outside...this process, we had an idea that survived the literature review, survived technical feasibility analysis... fearful of this technology and angry at the agriculture companies that produce GMO food plants. When ...idea, we knew it was a winner – although we didn’t figure out how we would accomplish the color change until...science, and to have a job that we enjoy and see a future in. And we decided to make education and outreach...

...not based on any existing protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize...characterized it, producing crystal structures demonstrating “the structure is strikingly similar to the design...works and creating biological tools not found in nature. Dr. Baker’s team was the first to develop and ...wanted to create a new fold that is not present in nature (at least that we’ve seen so far!). They started...the biophysics of protein folding and selective pressures during protein evolution. The Baker Lab has deposited...

... like to make sure they are present at a very low concentration. So how do we make sure to catch all of...experiment because of poor reagent quality. To make sure our AAV vectors are of the highest quality, we undertake...mission of accelerating scientific research. To make sure that adeno-associated viral (AAV) vectors available...all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...plasmid #44362. Method validation, limitations, and future plans How did AAV-68544 become contaminated? Well...processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544... the Addgene blog Check out our Viral Vectors Featured Topic Page Subscribe to Viral Vectors Blog Posts...

...antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  But llama immunization...proteins that may unfold due to camelid high body temperature (37.2 - 39.2°C). The Seeger lab found a way around...protein targets, similar to how antibodies do. The structure of the common antibody is heftier (~150 kDa) and...molecular crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...protocol, which cover 3 naturally-occurring nanobody structures (concave, loop, and convex). Each of these 3 ... to select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies...anti-Myc antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target...

...to Our Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9...of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent Cas9...alterations observed at the target site.   Figure 3: Gene repression with nuclease-competent Cas9... systems. She is also a NASA Fellow studying biosignature preservation in Mars analogue environments. ...proteins for RNA-guided gene regulation and editing." Nature methods 10.11 (2013): 1116-1121. PubMed PMID: 24076762... parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed PMID: 26167643...for genome editing, activation and repression." Nature methods 12.11 (2015): 1051-1054. PubMed PMID: 26344044...

...situations in cells are described below (See Figure 1).   Figure 1: Each problem with dimerizing fluorescent...follow. It's a matter of concentration Most FPs in nature are prone to dimerization (i.e. EGFP) [1, 2] or...together and can wrap the membranes into stacked structures including organized smooth endoplasmic reticulum...state, and an artificially active state due to closure by FP dimerization.   1. Transmembrane fluorescent...if a dimerizing FP is fused to the POI. These structures are large (microns), bright, non-physiological...whorl formation assay, are practical real world measures of dimerization potential that will determine ...GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429-...

...10hours. Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For...no use to you. When using more than one FP, make sure their emission light does not overlap in wavelength...new GFP isolated from the Unagi eel was shown to mature independently of oxygen, making suitble for use...studies (24-30°C). Brightness: Brightness is a measure of how bright is the emission. Brightness is calculated...parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent molecules gets ...lose the ability to emit light) after prolonged exposure to excitation light. Photostability can be as ...parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter is important if...

...scientific community and their game changing nature. Full disclosure: Zimmer also spends some time on Addgene...Zimmer’s recent book, The State of Science: What the Future Holds and the Scientists Making It Happen, is an...I began my own career over 20 years ago, and I’m sure they were issues long before then. Other areas are...for me. A brief window in time “The most obvious future consequence of the undermining of the scientific...and technology.” This last section of the book, Future Science, describes the author’s thoughts on science... science and summarizing its past, present, and future in under 200 pages is an impressive task to set...Science could be a jumping off point to explore future rabbit holes. For the non-scientists in your life...

...inhibitors to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove ...distance. Homology arms and types of donors To make sure your donor molecule is used as the template, you...your Cas9 cuts in the coding region of a gene, make sure that the PAM edit you introduce is a silent mutation...exist as a repair template. To maximize HDR events, ensure cells are actively cycling through the cell cycle..., they will not undergo DNA replication, so make sure you are keeping an eye on those cells! To take this...mediates efficient homology-directed repair and termperature-controlled genome editing. Nat. Com., 8(2024...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about Non-Homologous End Joining...

...different systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown... interest(POI’s) are fused to TAs and DBDs (see Figure 2a) (4). These fusions are then co-expressed in...DBD-POI fusion (commonly referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on...promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection of...the HIS3 gene, which is required for histidine manufacture and therefore growth on his-deficient media. ...known to bind serves as a good positive control to ensure that your setup works. The last thing you want ... system to detect protein-protein interactions. Nature. 1989; 340:245-246. PubMed PMID: 2547163. 5. Ozawa...