...pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing replication factors...outcome and planning.  Both ddPCR- and qPCR-based titering assays measure physical titers, meaning the concentration...those will quantify both genome-containing (full) and non-genome-containing (empty) capsids, thus leading...TCID50 measurement reports an infectious titer, meaning the concentration of viral particles that can transduce...concentration of viral particles containing a viral genome. While ddPCR is superior in precision and variability...accessibility of instruments in standard labs. When determining titers by qPCR, we recommend using primers targeting...

... be as pure as for other uses.   Cloning grade DNA Molecular cloning includes a set of techniques that...extraction. Figure 2: Use cloning grade DNA for direct cloning of your gene of interest into a ...in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure ...can have a plasmid or viral origin. The type of cloning approach you choose ultimately will influence the...DNA (ng range). To allow efficient and flawless cloning it is important that the preparation is pure from...the Addgene blog Browse blog posts on plasmid cloning Read our Plasmids 101 blog posts Learn about the...

...professionals is learning to succeed in grants through trial and error, through winning and losing funding...may consider earning a degree in Grant Writing, Management and Evaluation and/or earning the GPC credential...grant writer’, but make no mistake, researching, winning, and effectively managing grants involves much ...consulting firm, a Twitter Chat, and then our grant training and mentoring. How do I become a grant professional...to help you enter and to have a successful and meaningful career in the grants profession: 1. Build on ...There are many grant consultants who offer grant training, mentoring and coaching services, including Smartegrants...entering the Grants Profession. Whether you’re transitioning from a previous career or you’re starting your...

...Scientists enjoy learning new things. Building new relationships is all about learning new things from ...Training as a scientist in the academic system has many pluses. I delighted in my graduate school years...nature of academia often means that scientists-in-training rarely get the opportunity to learn about the ...science. I find it ironic that we do all of our training as scientists (5-12 years worth!) with academic... one of the best things you can do during your training is meet interesting people doing interesting things...Informational Interview it is understood to be a nonthreatening request to discuss experience, interests and...really, always). Informational Interviews are for learning and for expanding your web of connections. A mistake...

... explore beyond the expected. By widening your search and maintaining an openness to unexpected possibilities...simple steps to welcome these unforeseen moments. Widening your search The first step towards inviting serendipity...ultimately enhancing your project's potential. Widening your search creates fertile ground for serendipitous...could spark unforeseen connections and ideas. Maintaining openness and looseness of spirit The second crucial...element in harnessing the power of serendipity is maintaining an openness and looseness of spirit. Amidst the...of inspiration. Additionally, journaling in the morning has proven invaluable for me, allowing me to pinpoint...think beyond the confines of a predefined path, opening doors to novel ideas and approaches. Moreover, ...

... multiple cloning site to insert YGOI, a bacterial selection cassette for growth and cloning in bacteria...conditions. The first thing you'll need to decide when running your experiment, is which vector will best suit...bells and whistles to your construct. If you are planning to track your protein in the cell or purify it...expression vectors are inducible (e.g. pBAD LIC cloning vector (8A)) and have an epitope tag (6xHis or ...expressed protein with a specific resin (e.g. 6xHis containing pDEST-HisMBP). After purification the protein...pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check out our complete guide to empty ... Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other Plasmid Protocols List of common ...

...option to indicate separate promoters, tags, or cloning information for each insert.  Encodes gRNA/shRNA...sequence. Empty backbone A plasmid designed for cloning a gene/insert into it. Empty backbones often contain...proteins that are appended to new inserts through cloning. *Note — If you will be depositing multiple plasmids... information about the gene/insert(s). Step 3: Cloning Information Please enter the name of the vector...vector backbone information. List any promoters, cloning details for how your gene was inserted into the...for each insert individually.   Figure 9: Add cloning information for the gene or insert. Step 4: Growth...possible, we propagate plasmids in the standard cloning strain DH5α. Please indicate if your plasmid cannot...

...my live-dead staining. (B) Changing the y-axis from SSC to FSC can alter the positioning and shape of ...After several steps of tissue processing prior to running my flow analysis, I can however clearly identify...’s always good to ask around for advice before running your analysis. In this case, I and other lab members...and other larger cells. Note, we don’t mind the warning message at the top, as our cells of interest do...ones (Figure 3). A more “liberal” gate in the beginning should not have a great impact on your analysis...gating pipeline, it might be worth going back and loosening the FSC/SSC gate.   Single-cell gating Once you...duplicates can’t be reliably analyzed on their staining signals. For this, you’ll need to gate based on...

...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...unknown sequence with restriction enzymes and then running the products on an agarose gel allows a researcher...Labs still keep restriction enzymes on hand for cloning and analytical digests. A researcher can test the...dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string of DNA (...sequencing, such as confirming the results of a cloning experiment, require only one or a few sequencing...

... fall under informal learning; however, many others partner with formal learning to engage students at...environment such as the classroom or a defined learning program. Learners cannot (theoretically, at least...are interested in breeding dogs who also enjoy listening to podcasts A series of Tweets breaking down...caregiver communities  Table 1: Examples of defining your audience for science communication activities...activities  Many people are concerned that by defining a specific audience, they’ll exclude others from accessing... to you and a few other people on your team?)  Defining your audience allows you to create common ground...re ready to get into the more exciting parts - learning more about science communication itself! See you...

...predictable cut site) to develop the first plasmid “cloning” experiment. Dr. Cohen and colleagues treated a...proved successful, pSC101 became the first plasmid cloning vector and molecular biology was never the same...mammalian) species were cloned into plasmids and new cloning vectors such as pBR322, pACYC, and pUC were developed...copy number vectors that could be used in these cloning experiments. Although plasmids started as a somewhat...and recombination, replication and topology, and cloning and gene expression. Just released: Addgene'...eBook include: new chapters on genome engineering, cloning, and plasmid tags, addtional content added to the... Learn more about the history of plasmids and cloning: CSHL Meeting: Plasmids: History & Biology The ...

...validated. The creativity in employing a variety of cloning techniques and troubleshooting methodologies drew...colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation pipeline. (B) Schematic...the research community.    Validation For most cloning projects, the next step would be to validate your...which was observed for the hybridoma mAb.  While screening by IF-ICC worked well for identification of the...performing a BciVI digest of the PCR products prior to cloning, which in most cases left the functional light ...Approximately 70% of mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass ...collection References Andrews NP, Boeckman JX, Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, ...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic ...mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR... is an alternative method that can be used for cloning or genome engineering and is based on homologous...that differ from traditional restriction enzyme cloning will be explained in greater detail. Substrate... and can be increased to as high as 25-50% by designing oligos that avoid activating the methyl -directed...

...allencell.org and the Allen Institute website at alleninstitute.org. A classic challenge in cell biology is making...will add more to the collection later this year. Designing the donor plasmid Figure 2: Donor plasmid...make a precise cut in the genome near either the beginning or end of the gene of interest —in our case in... without outside influence (non-homologous end joining, or NHEJ), in approximately one percent of cases... process. By flooding the cell with a plasmid containing regions of high homology to the gene with the... relying instead on microhomology-mediated end-joining (MMEJ). To find out more about gene edited hiPS...allencell.org and the Allen Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic...

... but educators face significant challenges in designing suitable projects. In the O'Connor lab, we recognized... about the evolution of the genes involved in methionine synthesis (5). The budding yeast, Saccharomyces...-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae (Figure 1) occurs ... transformed strain to grow in the absence of methionine (i.e. the plasmid complements the deletion). ...enable multiple student experiments We used TOPO cloning to insert the coding sequences for ten different...plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to...strains on media that lack uracil, but contain methionine. (This allows cells carrying nonfunctional as...

...check out the distance learning courses at the National Council for the Training of Journalists (NCTJ) ...guided tours to show people where the lab magic is happening. 3) Volunteer – Step into science education Do...Learn on the job Nothing prepares you better than learning on the job. Try to get an internship at a magazine...internships to cover their annual meetings. 8) Training – Get a formal education Some people get into ...engagement for institutions and the Royal Society gives training in media skills, writing about your research and...moment, I got active with it, new doors started opening up. My current job as a Marie Curie Fellow is a... Additional Resources on Addgene.org Find Job Openings at Addgene ...

...back in 2007, which function as centres for peer learning and knowledge sharing, in the form of workshops...maintains a list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One...variety of aspects from the molecular biology to learning how to grow and care for the worms. As an example...relies upon biologists and computer scientists learning and communicating with one another.      Currently...declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00 a strain...optimizing the health of domesticated cats and learning about feline microbiomes. One of the aspects of...the kittybiome project, but the primary goal is learning in this case.   Funding Makerspaces One of the...

...requirements and expression limitations may be too constraining for some experiments, particularly in vivo ones...mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo CRISPR screens, AAV is ... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or... If you don’t feel like doubling the amount of cloning, another option is to use variants of Cas9, like...immunotherapy in glioblastoma, and generate programmable screening platforms for mouse tissues. Your AAVancement ..., Palop, J. J., & Kampmann, M. (2023). CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly...highly scalable cell type-specificin vivoscreening platform. bioRxiv (Cold Spring Harbor Laboratory). ...

...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...about management skills during your scientific training. I never thought about management skills before...grad school. Both were hard-working scientists-in-training who got the job done, but I struggled with what... he graduated. Years later I attended a formal training for new managers and I had a “Eureka” moment while... people! More specifically, they had different learning styles. One was a “Doer” and the other was a “...different levels of experience), find out what their learning styles are and capitalize on them for the benefit...it so shouldn't it be faster to do it yourself? Training certainly takes time - so plan accordingly -  ...

...Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury...already use. It makes the switch to Golden Gate Cloning easier because individual labs will not need to...make it easier for people to use the Golden Gate cloning system especially with plants in mind. When it’...There is no PCR, no gel purification; the whole cloning process is so simplistic once you have your parts... 30 constructs. They’ve never done any kind of cloning and they don’t have any prejudice about it. They... Sometimes it’s a good way to find out what’s happening in the office next to you. There is an active ... biology.  References: A Golden Gate Modular Cloning Toolbox for Plants. Carola Engler, Mark Youles,...