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...their biological materials during funding transitions. Enabling resource sharing: Our operating model allows...agility as a nonprofit resource can prove to be vital during this period of constrained funding. Addgene is ...lost amid lab closures or project terminations, ensuring that scientific progress made through years of...structures and indirect cost caps more efficiently. Offering free educational resources: Our eBooks, videos...productivity and efficiency with fewer resources. Fostering new partnerships: We're seeking and expanding ...strengthening our commitment to collaboration and resource sharing, we can navigate this difficult period together...what support would be most valuable from Addgene during this time. Your input will directly shape our response...

...consideration when titering a lentivirus is the cell line that is used for titering. Typically researchers... my (lentiviral) generation The first step in delivering genes via lentivirus is deciding what packaging... competence can arise from recombination events during viral production and poses a biosafety risk to ... plasmids in order to obtain the best titers. Requiring an additional plasmid for transfection can make... are required to generate RCL. To overcome the titering hurdles of third generation packaging systems,...will choose a relatively permissive cell line for titering in order to obtain the best titer possible. While... One should also consider the specifics of the titering transduction experiment. Media choice, reaction...

... and morphology, rendering their continued use inadvisable for experiments requiring reproducible results...visible and you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for...remove all jewellery as well as that watch you’re wearing before you start. Also, avoid unnecessary talking...hood. Planning your experiments thoroughly before entering the hood is another great way to prevent contamination...you’ll know exactly what materials you’ll need to bring into the hood and can wipe them all down before ...air flow around your work area. Make sure to only bring required materials into the hood. This will give...sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay out too...

...accomplish is to make the engineering of biology much easier. So you could imagine engineering the entirety of ...Stanford develops engineered DNA systems capable of storing data and computing inside living cells. He's also...an organization working to advance biological engineering openly so as to benefit all people and the planet... what we think of as an operating system for engineering an entire cell. Typically when people use plasmids...cell. Until we get a handle on precise, forward engineering of the central dogma at the genome scale, it'...collection of genetic parts for precisely forward-engineering the central dogma across many genes simultaneously...been 40 years since the invention of genetic engineering. What's incredible is the bioeconomy is already...

... generated will enter the culture medium during subculturing processes or from the air if the cell line...that are generated (while talking or pipetting) during working in the laminar airflow are also among the...source of the problem is the media, used for subculturing, which is available in both liquid and powdered... before sampling (Uphoff and Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard...separate lab should be made available for cell culturing and maintenance of the cell lines. Providing such...coats. Have separate footwear for the lab to avoid bringing in environmental contaminants.  Figure...Addgenie works in the tissue culture hood while wearing PPE. Photo by Kate Harten DeMaio. Cell lines...

... cell or neuron type may not work well for a neighboring cell type, even within the same brain region ...What AAV vector is best for my specific system?  Sharing data and community concerns AAV vectors are complex...particular importance to these efforts is the open sharing of negative data.  Negative AAV data is highly ...why they don’t share, many express concern about sharing negative data they cannot explain, perhaps out ...therefore actively work to remove the stigma around sharing negative data and instead focus on getting this...if many scientists choose to help each other by sharing their data. So take advantage of it and get your...Lukner, H., Kogermann, K., & Sepp, T. (2021). Data sharing practices and data availability upon request differ...

... January 2025.  What will the ordering process look like?  The ordering process is split into two steps...   Figure 1: The process of requesting and ordering a Packaged on Request viral vector prep.  ... research and expanded choice when it comes to ordering high-quality materials from the Addgene repository...Retrograde (AAVrg) capsids.  Please be aware that this ordering process will look slightly different from our ...steps, requesting and ordering. This split allows us to provide researchers with increased choice and flexibility...flexibility; while ensuring we can maintain high standards for both our depositors and requestors.  Requesting.... There is no charge for the request process.  Ordering If your request is approved, you will receive ...

...without the undesirable side products generated during chemical synthesis of these molecules. To make ...recombinant protein purification and metabolic engineering. If instead you want to build genetic circuits...biology MoClo applications, but there are more! Engineering disease resistance through gene stacking, increasing...kit has a focus on transformation and genome engineering. This is a specialized system with parts including...Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology...Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. 2017. ...Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes.” The Plant Genome, vol. 16,...

... she's either day-dreaming about exploring sci-fi worlds or exploring nature in the real world. Additional...ICE on Synthego’s blog. CRISPR-based genome engineering has revolutionized the gene editing field by ...edits performed over multiple experiments and comparing the robustness, accuracy, and speed with existing... of the sample name. If there was a minor error during processing, the window shows a yellow checked circle...with non-wild type sequence) as determined by comparing the edited trace to the control trace. In the ...Value - When the ICE linear regression is computed during generation of the ICE Score, the Pearson correlation...sizes in the entire edited population of genomes. Hovering over each bar of the indel plot shows the size...

... Figure 1: Addgene’s Agile Approach Sharing is caring There are many moving parts to our software...overall soundness of the software design. Each of us brings slightly different cognitive biases as a result...defined chunks, called stories, and work on stories during short periods of time called sprints. The team ...different technical approaches that may come up during discussion.  One example of agility at work is ...we recently reduced the number of steps involved during checkout. Demo details for this would be something...diversifying our own knowledge of the software, sharing knowledge allows for life to happen without impacting...developing new features and fixing bugs: we take time during each sprint to do some maintenance work on our ...

...Wisconsin lab for a summer during my PhD and is responsible for my enduring love of cheese curds. We’...long line for bag check was definitely worth it! Hearing about his passion for global health and the various...one of the highlights of the conference. It was inspiring to see how microbiology research is improving ...researchers who weren’t aware of Addgene’s plasmid sharing services, as well as #happydepositors who stopped...leverage their expertise as we advance our mission of sharing science. I got to check out many of the posters...about our mission of open science and resource sharing and was also a great place to catch up with former...distribution service! Did you see a must-have plasmid during ASM? Let us know what’s missing in the collection...

...combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology. pMA7CR... a backbone-targeting gRNA cassette for plasmid curing through "self-destruction" after induction with...CRMAGE is much more efficient than traditional recombineering, with 96-99% efficiency for point mutations...for a single round of editing, and a subsequent curing protocol that requires only 2-3 hours incubation...References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic chromosomal rearrangements with ..., et al. “Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.” Nucleic Acids.... Sakuma, Tetsushi, et al. “Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector...

...repair and genome engineering CRISPR-Cas9 has revolutionized the genome engineering world and made targeted...Originally written by Marcy Patrick and Mary Gearing on Mar 12, 2015; updated by Christina Mork, Jul ... fill in the resulting ssDNA. A single HJ forms during this process and resolves by cleavage of the crossed...immediately applicable in DSB-induced genome engineering, it has biological importance for the repair ...intact. In other words, Cas9 will keep cutting and repairing your target DNA until a mutation is introduced...Homologous Recombination. Jasin M and Rothstein R. Cold Spring Harb Perspect Biol (2013). PubMed. DNA double-strand... (2018). Pubmed. CRISPR References: Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. ...

...other to be physically present at certain times during the day? We asked three lab heads how they go about... If you are looking to join a lab, you may be wondering “What is this PI looking for in a new researcher...of CVs collected from throughout the year since hiring opportunities arise at various times. If the timing...will provide a solid foundation on which to thrive during time spent as a graduate student or postdoc. Although...chosen research field is important, the end goal during this time is to be trained to think and execute.... Or perhaps, the lab has a reputation for not entering collaborations. Alternatively, perhaps the lab...to ask lab heads, lab members and lab neighbors during the interview process. What if I want a non-academic...

...some of these questions in the sections below. Mentoring relationships can help identify your priorities...Alumni meetings can be the most informative way of gathering data. Former lab members can give open, revealing... Know who's boss You sign on to a lab for the mentoring only to find out that the lab head is an absentee...You are not allowed to speak to lab members alone during the interview period. People seem to be hiding ...about discussing data. There’s hesitation before answering questions about lab culture. Support for diverse...ensure that you are expanding your options and preparing for both academic and non-academic career paths...evidence of industry collaboration? If you are considering a career at a small, teaching college you might...

...methods for physical shearing exist as well, such as sonication and needle shearing. Essentially, any method... with the lab during its recent move to the University of Georgia). She enjoys pondering how phages get...between 1 and 3 kb results in optimal likelihood of capturing a whole RBP gene without causing issues associated...following colony PCR of 10-20 colonies using primers bordering the multiple cloning site of the vector. In order...saturate each with lysis solution (~3 ml each), pouring off any excess. When your protein induction is .... Leaving the membrane overnight in the freezer during one of these steps is also a convenient stopping...through the day, spending time with her horse, and wondering what her cat is thinking. Find her on Twitter ...

...for many metabolic engineering and synthetic biology goals. In metabolic engineering, the expression of...interfere with the precision needed in metabolic engineering or other synthetic biology circuits. What is ... a look at how the stabilized promoters perform during these types of fluctuations. When cells are dividing... can also cause changes in plasmid copy number. During the shift from exponential to stationary phase,...promoters were able to better modulate gene expression during shifts in growth rate as compared to a constitutive...To simulate the increase in plasmid copy number during the transition to stationary phase, Segall-Shapiro...they could alter copy number of the plasmid by altering IPTG concentrations. Higher IPTG concentration...

...leads to incomplete reprogramming. Additionally, lingering expression or re-expression of viral transgenes...temperature sensitive Sendai virus which is removed by culturing cells at 38℃, but screening cells for the presence...considered the safest since there’s minimal risk of lingering expression of reprogramming factors. Non-replicating...the plasmid in mammalian cells and assists in tethering the vector to the cell’s chromosomes. With the... high marks for safety.   Protein Delivery: Delivering reprogramming factors as protein is another way... week for 6 weeks, and still required further culturing to generate iPSC colonies. Reprogramming efficiencies...three germ layers (ectoderm, mesoderm, endoderm). Measuring pluripotency of mouse cells typically involves...

...makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots... the growing PubMed article list, the Genome Engineering Using CRISPR/Cas Systems has expanded out of ...wildtype or double nickase for my CRISPR genome engineering experiments? A1: When assessing which nickase...nickase type to use for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized...amazing resources for the scientific community and sharing these resources with Addgene. Thank you, Le! This...K., & Carroll, D. (2013). Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases. G3 Genes... L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/CAS systems. Science, 339(6121),...

... organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us ...laborious handling (screening 768 F1 animals). Recovering rare editing events in a sea of unedited animals..., which makes screening extremely rapid. This stringent selection allowed me to optimize a range of editing...al., 2018). They further built on this work, discovering that melting and rapidly cooling dsDNA repair...also found that the majority of knock-ins were occurring 24 hours post-injection, making screening for ...where the 5’ ends of the PAMs are facing outwards, ensuring the PAMs remain on the chromosome after DSB formation...Toolkit and SapTrap-SEC kit for C. elegans genome engineering have also facilitated CRISPR editing in C. elegans...