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...genome engineering workhorse You rarely hear the word ‘CRISPR’ without shortly thereafter hearing ‘Cas9...Cas family of proteins has many useful genome engineering enzymes, each with their own unique features ... and quirks. Here, we will review the genome engineering abilities of the lesser-known Cas enzymes while...Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good reason. It is guided by an RNA...strand break. Cas9 has been studied for genome engineering purposes far more than any other Cas family protein...making Cas9 an adaptable choice for many genome engineering endeavors. For more information on Cas9 variants...particularly convenient for targeting experiments requiring single strand DNA generation, such as homology...

...presentations and a handout on protocol sharing, material sharing, computational reproducibility, and tools...wet lab protocols, reagent sharing, bioinformatic tools, data and code sharing, and data analysis and visualization...identify the right opportunities, he was able to bring together talented and motivated groups and individuals...s contributions by Tyler Ford included reagent sharing and were presented at the ASPB meeting by the collaborators... exclude different modules for this workshop. Empowering others to have the materials and resources to... everything that you would want to know before, during, and after organizing such a workshop. Finally,...Finally, the group put a lot of effort into ensuring that the content of the materials and the workshops themselves...

... and Laura O'Regan Ordering jeans online from your favorite store is easy. Ordering genes on plasmids ...to other ordering questions here. In addition to answers to common questions about the ordering process...most frequently asked questions related to the ordering process: I placed an order through my university...will be able to enter your PO number as payment during checkout. Placing your order online has some benefits...Add all your items to your cart. Click checkout. During the payment step of checkout, you will have the... invoice to the billing address that you listed during checkout. This invoice will reference your Addgene...Addgene order number and PO number that you entered during checkout. At this time, your organization will ...

...approved 91,023 during the first 3 quarters of 2017. As an international researcher and aspiring entrepreneur... has been a growing shift away from employers sponsoring individuals for green cards because of the cost...have different roles, titles, and responsibilities during their careers - being aware of this can help take...regulations define a "specialty occupation" as requiring theoretical and practical application of a body...limited to biotechnology, chemistry, architecture, engineering, mathematics, physical sciences, social sciences...business specialties, theology, and the arts, and requiring the attainment of a bachelor's degree or its equivalent...work-authorization is strictly limited to employment by the sponsoring employer. On April 18, 2017, President Trump ...

...vector into the culture medium during adeno-associated virus manufacturing. Human Gene Therapy, 21(10), ... iodixanol) using a high-speed ultracentrifuge. During the spin, the virions will form a band due to their...check out the Addgene AAV Production protocol. Titering Once you’ve produced, harvested, and purified ...titer of your viral vector solution. A variety of titering methods have been developed to determine the titers...TCID50) (Zen et al., 2004).  To choose the optimal titering assay, you’ll need to understand how different...outcome and planning.  Both ddPCR- and qPCR-based titering assays measure physical titers, meaning the concentration...has to be digested with DNase before undergoing titering, to remove residual plasmid DNA carried over from...

...worry about traveling during an already stressful time, and I was more relaxed during both sets of interviews...yourself to a few members of your desired department during the application process. Many institutions recommend...in case one isn’t able to respond to your email. During my PhD application cycle, I reached out to around... and make sure you cover everything you need to during the interview. (Virtual) Campus Visits After receiving... financial and logistical obstacles could make touring potential programs and universities stressful or...Through the virtual systems created and promoted during COVID-19, online meetings with current students...worked with students to maintain these time goals during COVID-19? How are students funded? Are there any...

...genome engineering across multiple species Recent discoveries in the fields of genome engineering and synthetic...multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...gene of interest. Although the parts involved in altering gene expression are relatively well known, particularly...labs who wish to harness this powerful genome engineering tool. Bassett, et al. J Genet Genomics. 2015...loci (multiplexing). Currently, the only genome engineering method in bacteria that enables rapid, automated...combinatorial genomic libraries. MAGE has allowed genome-engineering endeavours of unparalleled complexity in Escherichia...mutant enables transient suppression of DNA repair during oligonucleotide integration, allowing MAGE in otherwise...

... with limited time for one-on-one mentoring. His solution? Bringing his research into the classroom. I...textbooks” (Brian, 1996). But is it possible to bring the kinds of transformative educational experiences...dehydrogenase (MDH), a citric acid cycle enzyme he studied during graduate school. As a stable, easily purified protein...interest to other researchers in their field. Indeed, sharing results with the broader scientific community represents...all shaped his teaching style, with the former offering excellent training in improvisation and the latter...Jessica Bell, Lisa Gentile, Ellis Bell, and Amy Springer), this group now connects MDH-focused CUREs at...available technical and pedagogical resources, offers mentoring for instructors, and provides opportunities for...

...community. Sharing is caring We’re thrilled that our depositors reap the benefits of sharing plasmids with...hooked on sharing because, as UNC’s Bryan Roth says, Addgene has “literally enabled sharing with 100's...plasmids deposited at Addgene enhance scientific sharing worldwide but a select few are particularly special...honor the creativity, passion, and dedication to sharing epitomized by the creators of these plasmids with...appreciate our depositor’s dedication to scientific sharing, we wanted to celebrate our depositing PI’s who...deposited in Addgene!” As you can imagine, answering plasmid requests can be a time-consuming chore...Flame Award winners- we’d like to thank you for sharing your plasmids at Addgene. You are the reason we...

...mix. During the incubation the immobilized antibody binds to the target protein, thereby tethering it to...approaches allow for optimized strategies for differing amounts of target proteins or antibody bonding...immunoprecipitation and simply refers to the process of anchoring an antibody, often called the capture antibody...eliminating the risk of the primary antibody interfering with downstream assays. This is particularly ...for an IP, samples are typically lysed in a non-denaturing buffer containing non-ionic detergents, such ...proteins, including those in the nucleus, more stringent buffers may be needed. Since many factors, including...Frequently, the antibodies used for a standard, denaturing Western blot will not be suitable for an IP, ...

...team building and bring back some degrees of belonging that was probably lost during these weird times...Discussing data with colleagues can be more difficult during the pandemic era, with fewer opportunities for ...   Implement new tools for creative interaction During Zoom meetings, different tools can be used to promote...The annotate function can also make the screen-sharing experience more interactive. The button is on the...collaborative cloud tools such as Google Docs and Sheets during Zoom lab meetings to chip in ideas and take minutes.... This document can be shared and serve as a springboard for future discussion. Collaboration and creativity...great functionality that is widely used and could bring back a taste of that casual kitchen chatter, ultimately...

...immune system to fight progressing cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer... are coming together to tackle the challenge of curing cancer in cross-disciplinary projects and collaborations...reflect how cancer/immunology researchers are preparing and carrying out this multi-pronged attack. Inflammation...our recent post “Cancer and the Immune System: Deciphering the Relationship” by our guest blogger Subhadra...CRISPR/Cas9 methodology with T cell receptor engineering (Ref 5)  as described by Blue Flame Addgene depositor...Professor Michel Sadelain from the Memorial Sloan Kettering Cancer Center. In contrast to using randomly integrating..., CRISPR/Cas9 genome editing allows targeted engineering of the TCR locus. He also highlighted the UCSF...

...to all of our generous, sharing depositors. Over 3,000 laboratories are sharing plasmids (with 16 labs ...labs currently sharing viral preps) through the Addgene repository. Without these scientists and the support...to have such an impact on scientific materials sharing. I would also like to recognize a few scientists...Addgene has been successful in helping thousands of sharing scientists from laboratories all over the world...Scientist Team has been developing expertise in answering questions on virus and helping provide educational...reach scientists around the world. Our Software Engineering Team (and, added in 2018, our Product Team) continues...Maintaining our excellent service and accuracy involves caring and careful Addgenies, but also a lot of good tracking...

...the work by using the principles of recombineering. In recombineering, bacteria express phage recombination...REPLACR-mutagenesis, harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions...how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized Plasmids After PCR). ...modification by homologous recombination. In most recombineering strategies, only 3-4 phage proteins are needed...directly transformed into bacteria expressing recombineering proteins Red/ET from a temperature-sensitive...bacteria are subsequently grown at 37 °C, the recombineering plasmid is not maintained. Recombination efficiency... the need for selection usually required in recombineering. REPLACR-mutagenesis is also well suited for...

...into their scoring functions. Many tools have also opted to mix-and-match different scoring algorithms... by comparing their past results to the designs suggested by each tool. For those who are entering sgRNA...facilitate most experimental applications in CRISPR engineering. When compared to that collective utility, it... and approximate methods. The details of their scoring function can be found in their online tool’s documentation...is by far the most common with most tools also requiring that the user specify an organism. Output diversity... genome”, “large genome”, and “organism biased scoring”. Trends: Many tools can handle genomes of any ...solution but are often packaged as offline tools requiring expertise in one programming language or another...

...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...