We narrowed to 888 results for: TRI

...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, ...to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble... Gate protocols, complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet...destination vector contain multiple internal restriction sites that may not be amenable to "domestication...cloned DNA as tandem multimers using class-IIS restriction enzymes. Lee JH, Skowron PM, Rutkowska SM, Hong...

...unique restriction enzyme site and unique primer sites for more rapid screening by restriction digest ...for genetic optimization of industrial yeast strains. However, industrial yeast strains are more difficult...CRISPR library Addgene has had the privilege of distributing several human and murine CRISPR pooled libraries...single-tube, 3 day process by utilizing pre-assembled trimers to reduce the number of cloning steps. The FusX...PMID: 26787909 EasyClone 2.0 Yeast Toolkit Many industries, such as the food and beverage industry, the ...transform. To increase the genetic toolbox for industrial yeast strains, the Borodina Lab has recently ...et al. Nat. Methods 2009. PubMed PMID: 19915560 TRIBE: Targets of RNA-binding proteins identified by editing...

... on versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to ... But...how? Cloning is no longer limited to restriction enzymes - although many labs still love using...your E. coli do the work for you. Traditional restriction enzyme and ligase based cloning is a relatively...The ligase cost is comparable to that of the restriction enzymes and overall you’re looking at a price...of a few dollars per reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning...appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations. The...efficient due to the use of the versatile type II restriction enzymes. In Golden Gate cloning, the desired ...

...Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole field ...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...would take another 6 years before the 1st clinical trial started, and 10 more years to obtain FDA approval...natural serotypes are currently used in clinical trials, a variety of engineered ones have shown great ...in preclinical studies and could reach clinical trials soon. The AAV safety profile is excellent and ... cells using a lentiviral vector and a clinical trial is ongoing. Moving forward with ocular gene therapy...AAV-based ocular gene therapies is impressive: clinical trials for choroideremia, X-linked retinitis pigmentosa...

...This post was contributed by guest blogger Katrin Michel. Cre-lox is an incredibly popular and powerful...times. Many thanks to our guest blogger, Katrin Michel! Katrin Michel is a Postdoctoral Researcher at MIT...pair palindromic repeats separated by an 8 bp asymmetric core. Recombination between these sites can lead...discovered that it is not the exact sequence of the asymmetric target site core but its 8 bp length that is ...plasmids and the low concentration of recombinase restricts their expression to individual cells. With this...knockout in the same mouse.   Scientists always strive for more. In order to analyze the function of individual...Through a partnership with genOway, we are able to distribute materials containing FLEx technology. Read the...

...legal approvals so the plasmids are ready for distribution as soon as the paper is published, while giving...always have control over when your plasmids are distributed. Next month, we are making two changes to the...with permission to release their plasmids for distribution, and we’ve found that this step was sometimes... you on your paper!). And as always, we won’t distribute materials with unresolved sequencing issues or... deposit due to experimental problems, legal restrictions, or any other reason. But this way, as soon ... published we’ll get the plasmids online for distribution instead of risking leaving them on hold indefinitely...us to process and store materials that aren’t distributed or for our teams to keep searching, emailing,...

...level, read on for more in-depth tips on probe chemistries, primer optimization, controls, and analysis....Picking your chemistry  The two most common qPCR chemistries are hydrolysis probes (which you probably know...commercial name: Sybr). However, there are other chemistries available and they can be the more optimal choices...choices at times. For example, chemistries that rely on hairpin design of probes/primers often offer higher...stable than the hairpin bond.  Alternatively, chemistries that combine the primer and probe into a single...comparison of features of several commonly used chemistries)   Chemistry Background noise Probe ...gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design...

...studying the brain in a way that goes beyond such restrictions, you may want to consider a systemic capsid ...enhanced function after intravenous administration, distributing widely throughout the organism to target large...and recombination target sequences, denoted by triangles] facilitate tissue or cell type specificity. An...ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal ...your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb) for your...promoters and enhancers (Mich et al., 2021) can help restrict expression to your cell type of interest, while... with Cre-expressing transgenic mouse lines to restrict transgene expression to specific tissues and cell...

... a SmaI or XmaI restriction enzyme digest (the ITRs contain SmaI and XmaI restriction sites). Another ...these tools, I found myself writing AAV production tricks into the margins of my protocol. While these tips...recombination of duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary...ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of different concentrations...during AAV production is overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected...day. Refer to the AAV Iodixanol Gradient Ultracentrifugation Protocol for more details. Pro tips Watch...titer AAV. Do you have any AAV production tips or tricks? Please share them in the comments below! Special...

...This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such...limitations through the use of engineered Type IIS restriction enzymes and careful choice of junction sequences...involves the simultaneous activities of a Type IIS restriction enzyme such as BsaI, and a DNA ligase such as...receives the assembly after similar Type IIS restriction enzyme cutting results in 5’ four base overhangs... provide optimal reaction temps for both the restriction enzyme and ligase, thereby enhancing the ordered...destination construct and functioning Type IIS restriction enzyme and DNA ligase, any single omission should...

... This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means... assumed that the molecules are homogenously distributed throughout the volume and can freely tumble in...be much higher than if they are homogenously distributed and mobile throughout the cell. If two copies...CFP, YFP, BFP, Cerulean, Turquoise, Venus, and Citrine) can be monomerized with an A206K mutation [2].... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing the lowest...

...research by banking and distributing monoclonal antibodies at cost. We distribute over 65,000 antibody units...The following post was contributed by the Developmental Studies Hybridoma Bank. Antibodies are the most..., is a repository for monoclonal antibodies, distributing over 3,000 antibodies and associated hybridoma...the University of Iowa. The minimal fees from distribution funds our operation. Antibodies are glycoproteins...PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies against popular fluorescent...Posts about Scientific Sharing: The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years...

... media platforms are available or used in all countries; not all areas of any given country have equal...can guarantee you that either resource or time restrictions (or both!) will be a major limiting factor at...flexibility to visualize and contextualize. Can be distributed in digital or analog media formats. Cognitively...platforms can splinter audience. Primarily digital distribution (though radio is possible.) Written works...works Low Flexible. Low barrier to entry. Ample distribution channels both digitally and physically. Easy...overwhelming your audience. Digital or print distribution.  Static. Can be difficult to convey an entire...highlighting, or omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific...

...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes...system (CNS). To address this technological gap, a trio of recent papers describe the development of CRISPR...method to dissect the individual and shared contributions of transcript isoforms to neuronal physiology...surface protein for which there are good flow cytometric antibodies, downstream of the TUJ1 coding sequence...these cell types are used. The lack of good flow cytometric antibodies for many CNS-resident cell types (...

...This post was contributed by Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...no one is suggesting all data be stored in a retrievable format, we too rarely preserve “raw” data of ... methodological detail. Publishers have also contributed to this issue by requiring ever more condensed...resources can offload the burden of data storage and distribution. I would argue that the journals themselves ...journal repository, they are likely too large to be retrieved by others. Other new data initiatives are also...and understand their data. Likewise, we want to contribute to helping others understand ours. Improving ...

... plasmids: Twitch sensors Griesbeck’s recent contribution to the field is a series of fluorescence resonance...which are available at Addgene, can be used for ratiometric in vivo imaging. Griesbeck explains, while these...both neuroscience and immunology. Don’t be shy: Ratiometric imaging for beginners Griesbeck says his calcium...beginners may have a tendency to shy away from ratiometric imaging, he recommends they give it a shot. “...practice optimizing the sensors involves plenty of trial and error. Transition metals also present challenges...dynamic range,” he says. Merkx recently applied this “trick” to the development of additional color variants...test.” References Thestrup et al. Optimized ratiometric calcium sensors for functional in vivo imaging...

...expert and the lack of credit for small contributions. We strive to eliminate these two barriers, creating...This post was contributed by Ignacio Amigo and María Hernández Sánchez from Crowdfight. In March 2020...between researcher and expert; 4) Credit all contributions.   Our volunteers are extremely diverse.... the project we are focusing on what we call “asymmetric collaborations.” These are transient partnerships...where a researcher makes a small but crucial contribution to someone else’s project. We think that these...A discussion about a protocol can save weeks of trial-and-error. A recommendation for the correct statistical...

... of times in 2 weeks. This is a striking example of the contributions that scientists are able to make... Figure 1: Biological repositories that distribute mouse strains, plasmids, cell lines, and much...Institutes of Biomedical Innovation, Health, and Nutrition in Japan. These results  will help researchers...investigation of this pathogen. The sharing and distribution of cell lines from repositories on opposite ...around the world. This scale of production and distribution is possible due to the dedicated facilities ...repositories’ capacity for storing, validating, and distributing such reagents are all ways in which the scientific...

...This post was contributed by Sreepadmanabh M, a member of the iGEM team from IISER Bhopal. Following ...The importance of psychrophilic enzymes From an industrial perspective, the enzymes obtained from psychrophiles...host The expression of recombinant proteins for industrial or research purposes requires stable host cells...either die out or grow so inefficiently that no industrial operation would ever consider them worth the ... Oleispira antarctica, and the P. haloplanktis Trigger Factor), that would allow the stable folding and...spirit with iGEM’s emphasis on holistic, community-centric science, our team has continuously engaged with...with a variety of stakeholders from industrial and commercial operators, government agencies, active researchers...

...initiative since we started distributing plasmids in 2004. We’ve already distributed over 500 viral samples...In the middle of 2016 Addgene started distributing a small but growing catalog of ready-made AAV and ...the infrastructure capabilities to produce and distribute virus. Addgene is a 501(c)3 nonprofit and our...lead on operationalizing viral production and distribution, this project involved every Addgenie in the... to enable efficient, tracked production and distribution of viral samples. The Office Team made sure ... plasmid depositors and their institutions to distribute viral preps derived from plasmids in the repository...