We narrowed to 1,042 results for: URE

...between strains of one species, so be sure to check the literature and validate your choice of AAV vector...Tropism depends on many variables, so be sure to check the literature for previous work in your experimental...adeno-associated virus infection. Nature, 530(7588), 108–112. https://doi.org/10.1038/nature16465 Tervo, D. G. R., Hwang...allowing them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...vectors have both distinctive capsids and genomic structures. The viral capsid is the key that lets AAVs “...mixing, matching, and modifying capsid and genomic structures. For example, rAAV pseudotypes mix the genome... by Addgene are pseudotypes using the genomic structure of AAV2, so they are formally called rAAV2/1, ...

... notes on how lab procedures were actually performed, potentially curtailing future confusion (and hours...community-editable fluorescent protein database ReFigure: Save Scientific Figures into Dashboards and Share Your Insights...reproducibility issues in the biological sciences and featured speakers from academia, industry, nonprofits, ...reproducible if its findings can be predictably applied to future work. Importantly, no one should expect that every...Failed attempts to apply irreproducible results to future work cost both time and money with estimates reaching...doing an experiment wrong. However, even simple procedures like pouring a plate or running a gel have intricacies...initiatives to create more reliable reagents for future research. I’ve talked to many researchers who have...

...factors to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup...activity or gene expression. Experimental output is measured and quantified. Created with BioRender.com. ...either excite or inhibit neuronal activity upon exposure to light within a specific range of wavelengths... be used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light...engineered receptor that change shape with light exposure to activate them. PORTLs use a similar concept...when a signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes...that may go awry in a disease state.      Figure 4: Example of an intracellular signaling pathway...

...10 minutes, but check with your enzyme’s manufacturer to ensure that you’re cutting for the proper duration... the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and...proper orientation. You then add ligase to the mixture to covalently link the backbone and insert and,... phosphatase) are commonly used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and...unfortunately you can’t just throw the digestion mixtures together. You need to isolate your insert and ...charged DNA through the gel. As indicated in the figure on the left, your digested DNA (and undigested ... the Bitesize Bio Blog and their associated manufacturers websites. These stains require you to either...

...which attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used...aeBlue, amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria...fluoresce at very low levels. Chromoproteins in nature Compared to the history of fluorescent proteins... chromoproteins lost color overnight in liquid culture, though expressing the chromoproteins from the ...chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and low fitness...     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple...genetic modification integrates into the genome (Figure 3). Here, the chromoprotein is expressed and gives...

...compositions of Cascade. Although seemingly ubiquitous in nature, they are largely underutilized in research settings...mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes... systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created...tools has been challenging. It can be difficult to ensure comparable expression of all components of Cascade...making cuts every so often. Cutting both strands ensures that there isn’t a template strand left behind ...behind that the cell can use to repair. Figure 3: General overview of the Cascade-Cas3 mechanism. Created...components can make Cas9 easier to work with than Cas3. Future targets Cas3 has the potential to be incredibly...

...MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced for future MagLOV-based...control of biological systems (Wheeler et al., 2016; Duret et al., 2019). But the field has sometimes seen ...1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and Maria were skeptical. ... since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in ...change of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was ...and didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a...magnet is waved under the sample. The imaged area measures approximately 82 mm across. Video courtesy of ...

... active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from...it integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top...However, the rate of transfer was variable. They measured transfer rates by comparing the number of colonies...several bacteria associated with human disease (S. aureus, Klebsiella pneumoniae, and P. aeruginosa) in addition...isolated from cheese. Knockdown efficiency, as measured by knockdown of a constitutely expressed RFP reporter...median knockdown of ~40-fold across all species measured. Beyond understanding the basic biology of bacteria...pathogens) were grown in presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to...

... resource. Nature Protocol Exchange Website: http://www.nature.com/protocolexchange/ Nature Protocol Exchange...for iOS and Android). A neat feature of protocols.io is the checklist feature which allows you to check ...process so you’ll have a record of your work. This feature further simplifies lab work if you like to bring...an open online protocol repository hosted by the Nature Publishing Group. Like the previously mentioned...and create lists of your favorite protocols for future reference. The exchange currently boasts over 2,500...links to protocols on their own websites. We're not sure how frequently it's being updated now, but there...access to protcols from external vendors, such as Nature Protcols and NEB. Quartzy Website: https://www....

...Martin, B., & Shendure, J. (2022). Precise genomic deletions using paired prime editing. Nature Biotechnology...editing world. The approach, originally published in Nature, is a “search-and-replace” genome editing technique... breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing. Created with BioRender.com...Liu lab contributed an improved prime editing “architecture” that can be combined with any of the PE2-PE5...prime editing as efficient as possible. Figure 2: Decision tree for selecting a PE6 variant. PE...2023, under a CC BY 4.0 license. epegRNAs: Structured RNAs with improved stability Traditional sgRNAs...of large DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. https://doi.org/...

...green cards and citizenship.   Lauren Celano, CEO Propel Careers. Lauren is passionate about working with...This post was contributed by guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. ...and on. As you think about your career (and your future in general), keep in mind that you may have years...careers - being aware of this can help take the pressure off having to find the perfect job immediately...but not limited to biotechnology, chemistry, architecture, engineering, mathematics, physical sciences...bachelor's degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...background, expertise, publications, and plan for future activities; including the interest of the greater...

... 1) follows this structure: Step 1: fix (and maybe label) your sample (i.e. cultured cells or tissues...and after expansion and measure the degree of agreement between them to ensure the expanded images are...how you interpret a target’s structure. For example, cytoskeletal structures that form continuous lines ...easier, more accessible way to image biological features previously only visible via techniques like super-resolution...by just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify...depend upon the specific gel recipe used, ambient temperatures, incubation times, and the physical properties...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain...

...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...will also efficiently ligate many undesirable structures, including substrates containing gaps or DNA ...phosphorylated base can be observed in the crystal structures of several DNA ligases bound to substrate (23... the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed.... 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction mechanism, and function." Chemical reviews...

....  After this, lowering the temperature allows the repressor to re-nature and again repress expression...red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering ...a generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering ... pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...few strains can also be found at Addgene, but be sure to read the associated papers for their particular...

...copies per cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how...Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum Freshly...colony and do not subculture directly from glycerol stocks, agar stabs, or liquid cultures. Freshly transformed...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...if you need two plasmids for an experiment, make sure they have "compatible" ori's. See the table below... affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...

... guest blogger Jack Leeming Editor of Naturejobs, part of Nature magazine. Humans are built to enjoy, ... your structure. Develop your protagonist In most cases, the protagonist will be you. Make sure to highlight...accept and acknowledge your failures. Foster conversation A story is not a lecture. Make yourself open to ... parts of our lives that tend to have a story structure: beginning, end; cause, effect; lesson, learning...apologised and admitted he’d been under a lot of pressure recently which had affected his judgement. Eventually...rhetorical technique you have in your arsenal to make sure people pay attention. Highlight your lowest points... discovery into a scientific story, but it will ensure that people pay attention. Practice Nobody gets...

...able to eliminate the need for axenic (pure) laboratory cultures to study these microbes. From 16S rRNA...GPS coordinates, water temperature readings using a heat gun, and pH measurements. We collected small water...samples could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery... bank and the vehicle battery/alternator to make sure the vehicle battery didn't run down. In September...processing environmental samples Since the high temperatures and unstable ground around these hot springs...-term stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot...able to safely get back to our lab work. Figure 3: When a bison wanders into the campsite, leave...

... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299 Find plasmids from...targeting with CRISPR–Cas13. Nature 550:280–284 . https://doi.org/10.1038/nature24049 Find plasmids from this...altered PAM specificities. Nature 523:481–485 . https://doi.org/10.1038/nature14592 Find plasmids from this...and high DNA specificity. Nature 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX,...Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) NGRRT or NGRRN Neisseria meningitidis...than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9...more about SaCas9 and AAV Check out our CRISPR Featured Topic Page Resources on Addgene.org Check out...

...preparations are added to cells in culture. Three to five days later, cell cultures are inspected for sterility...also performed by ddPCR.  Quality Control Addgene ensures high quality viral vectors by optimizing and standardizing...quantity of gram-negative bacterial endotoxin is ensured to be less than five endotoxin units per mL. The...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified...item (see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... darker than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation...packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first...

...There is no known safe level of exposure to asbestos – i.e., any exposure to asbestos is potentially capable...the exposure, the greater the risk of disease) there is no known safe threshold or “safe” exposure level... goal of the Cancer Moonshot Initiative being to cure cancer, more funding and research opportunities ...unfortunately, completely preventable. Mainly caused by exposure to asbestos, mesothelioma is a form of cancer ... not quick. It can take decades from the first exposure to asbestos to the time when someone starts seeing...average latency period (time from first asbestos exposure to the time of diagnosis) is about 35 years, but... has noted, there is no safe level of asbestos exposure. Anyone exposed is at risk of developing mesothelioma...

...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic...in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance...production either in the plant or in plant tissue cultures.   C. roseus is not a model organism such as Arabidopsis...used to develop stable hairy root lines in tissue culture. This method is commonly used in C. roseus, but...not reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables...overexpress a gene and evaluate its function. In nature Agrobacterium contains a plasmid with a T-DNA (... does not work well, as the leaves have a waxy texture and are difficult to infiltrate. We have recently...

... different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria...resistance is defined as microbial survival despite exposure to antibiotics designed to kill them or to impede...multi-drug resistance: definitions vary in the literature, but generally organisms with multi-drug resistance...contributing to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example...populations to antibiotics at unnecessary times, putting pressure on resistant bacteria to over-take the population... and making them more difficult to treat in the future. Patients not finishing the whole course of antibiotics...killed by a higher concentration, or sometimes more exposure time is needed for the drug to work… if the instructions...

...samples: What we did To measure the pH of our samples, we used a solution of cell culture medium, which is buffered...samples at room temperature and used the color of the liquid solution and pH paper to measure the pH. We compared...range of pH exposures. They showed that AAV infectivity was not altered by a short 2-hour exposure to pH as...-rings are, in fact, permeable to CO2 at low temperatures (-80°C) and once in the tube, the CO2 can alter...not all O-rings) were permeable to CO2 at low temperatures. We tested two different types of tubes: the...tubes after the indicated incubation periods, and measured the pH. We found that tubes subjected to a recovery... 2014). They also showed that even 24 hours of exposure to pH 5.1 did not alter the infectivity of the...

...pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down...using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry...contains the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started...protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which...using standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments...RNA (IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly...vectors or synthesize/purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags...

... the off-target activity. Figure 2: Specificity is the measure of off-target activity (left) while...sensitivity. Site accessibility Site accessibility is a measure of which genomic sequences can be targeted by the... sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...closely related to specificity. Sensitivity is a measure of the on-target activity of the Cas9 nuclease....frequency at a target site. A specificity ratio can be measured by calculating the on-target activity divided ...while sensitivity is the measure of on-target activity (right). Cas9 nucleases that exhibit enhanced...NGAN, NGNG, NGAG, and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9...

... adjustments I wanted to make to ensure smooth filming in the future, but we were finally refining the...I could be happy with it. Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being...trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for...anticipated, we had to think of something else.   Figure 2: Ironing boards. Good as a WFH desk, and as a...important for me to let people record at their leisure. For virtual B-Roll (that’s the secondary footage...drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social distancing... left the room. Microphone: Lastly, I needed to figure out a microphone situation. Generally, I record...

...extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (or...overlap ensures that the PCR generates a nicked circle rather than a linear product (see figure). Template... simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust the extension time to ...vivo to give the final circular plasmid product pictured here. B) Screening of recovered plasmids for the...final concentration). The DMSO reduces secondary structures of the DNA template, and may also decrease the...template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through...polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases...

...Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr...some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G... mouse model, an important proof of concept for future preclinical and clinical work. Find Plasmids for... is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV ...homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds...would be applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular...low-frequency indels will not cause frameshifts. Figure adapted from Osterout et al. (2015b) under a CC-BY...

... like pipets, or mini-centrifuges, etc. Be sure to capture as many of these tools as possible, this even... we suggest estimating the time it will take to secure your first (R-level) research grant.  Set your ...down prices as you create your list. This helps to ensure that all of the resources are accounted for. Write...an idea of how much you can expect to use in the future. 3. Identify the skills needed to use these tools...the talent pool that you’ll need to tap for your future research team. For example, if you’re going to ...help you define salary costs. 4. Ascribe a cost structure associated to these tools, skills, and personnel...searching for a faculty position. This is important to ensure that you’re looking at institutes that have the...

...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...