... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...design of flanking overhangs. Although there are theoretically 256 distinct flanking sequences, sequences that...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...

...undergraduate students (1, 2), but educators face significant challenges in designing suitable projects. In... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...functional MET genes to grow.) Students then use replica plating to determine if expression of MET/Met genes...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... Students who participate in the course show significant gains in both content knowledge and research ... research into undergraduate courses and in communicating science to the public. You can find out more...

...generated in vitro using synthetic genes that are typically expressed from a plasmid or from an integrated...cells, for antibody production and subsequent purification.  Academic laboratories and companies have already...share, making recombinant antibodies the most practical choice. Hybridomas are much tougher to ship, often... often lack sequence data, and may genetically drift over time. Antibodies made from animals are limited...the ease with which scientists can now improve genetically-encoded research tools, it is shocking that the...Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531–533 . https:/...Edition. 2104. Cold Spring Harbor Laboratory Publications, New York. Helma J, Cardoso MC, Muyldermans ...

...production and subsequent necessary modification of these chemical products. The flexible nature of MoClo... Gate cloning is MoClo. That’s because MoClo specifically refers to the organizational schema described...organization schema MoClo can be broken down into three hierarchical levels: individual DNA parts, transcriptional...together. They can express one or more gene(s), and typically begin with a promoter and end with a terminator...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...microbially or in cell-free systems. These pathways typically involve multiple steps in which a precursor molecule...genotype and phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You...

...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...researchers to identify and isolate very specific biological targets. However, as popular and useful antibodies...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...antibodies, creating a potentially unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively...a research assistant in lab of Zhuhao Wu at the Icahn School of Medicine at Mount Sinai.  References ...

...very useful in the event that you will not be physically present at your poster at all times, so other... sections should be as concise as possible, communicating only the most important results to your audience...have the advantage of being able to verbally communicate your work, so you don’t need every little detail...readers can navigate your poster and follow the logical progression of your work, and you can accomplish...offers free poster templates, although most are vertical.  Large central box. Another commonly used and...presentation. Portrait. This style displays a poster vertically rather than horizontally. Instead of displaying.... Shapes. In this type of poster, written and graphical information are inserted into larger circles or...

...Lacks - the African American woman whose cervical cancer cells became a mainstain of biological study without...advice on how to dive into new forms of science communication. Click here to listen to the interview with ...fossil rocks and their importance in understanding biological history. 4. Please Don't Kill My Hive Beautifully...implement the next generation science standards and communicate science. Learn more about Tom on his youtube ...

...example, if you’re a virologist presenting at the American Association for Cancer Research Annual Meeting...therapeutic strategies to target it, than you typically would at a microbiology meeting. Create an outline...lab mates, and PI can provide feedback on your technical presentation, but those outside of your field ...don’t fully understand the question, ask for clarification. Having them reframe or elaborate their thought... through the list at the session so you can strategically use your time. Don’t have access to a list of...titles to find the ones you’d like to revisit. Strategically traverse the sea of posters If you come prepared...system, and is interested in a career in science communication.       Resources on the Addgene blog Read other...

... more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction...stability, and buffer compatibility have placed practical limits on single- or two-step assemblies. We have...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence... While T4 DNA Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers...-Crick base pair substrates, it demonstrates significant activity on some mismatch-containing pairings... sequence-dependent ligation efficiency and identification of mismatch-prone pairings. Using these 4 bp...Kucera is a Principal Development Scientist of Applications & Product Development focusing on DNA cloning...

...recognized by its antibody without additional chemical modification steps and when the epitope was within the...antibodies for every potential new target and basically impossible if you wanted to distinguish between...C-terminus post-translationally, which turned out to be critical for antibody recognition. As such, the peptide...interest and proteins tagged with it needed to be chemically treated before the antibody could detect them... the N-terminal half of the antigen differs significantly between human and the other species, suggesting... flexible - it could now be used in way more applications.   Fig. 1 Making a Protein Tag (Abridged... of new versions of Myc1-9E10 mentioned above basically involve taking the Myc1-9E10 variable regions ...

... the pharmaceutical industry for years. They have proven very difficult to target therapeutically due ...al. "Systematic identification of signaling pathways with potential to confer anticancer drug resistance... millions of people die from cancer-related complications each year. Despite these grim statistics, new... cancers, and synthesizing compounds to bind specifically to their mutated residues. For Ras researchers...any of these genes through several different applications such as fluorescent protein fusion studies and...represented, making the results of any such study as clinically relevant as possible. Connecting the research...

...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...PMID: 26678082. Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015...633. PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei...

...transformative biological discoveries and life-saving medical breakthroughs and undermine critical research ...Addgene as a trusted partner for archiving their biological materials during funding transitions. Enabling...operating model allows researchers to access authenticated materials at a fraction of the cost of creating...be listed as direct costs from a CRO in grant applications, addressing the new funding structures and indirect...private partners to create additional pathways for critical scientific work to continue.  Moving Forward ...

... citations for recent articles. Of course, a publication in Science or Nature is always nice, but in the... lesser-known journal as it is in a top-tier publication. Accessibility is surely also behind the increase...increase in citations for older publications. (As an interesting aside, Nature recently reported that the...in a solution.) But are citations and journal publications really the best way to measure impact? As that...papers detailing Nobel Prize-winning research typically weren’t among the most cited. And, as scientific...and what it’s made of. Increasingly, journal publications alone aren’t enough. Scientists are sharing ...consider and quantify the impact of scientific publications. Ventures like Impact Story seek to find ways...

... and chemical contaminants that have been used during the purification procedure. Good indicators of DNA... is necessary for bacterial transformation of chemically and electrocompetent cells is minimal (picogram...nm should fall ~1.8 - 2 and give scientists an indication of purity versus protein contaminants. The 260...nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents as guanidine...addition to being free of any nucleic acid and chemical contaminants, these DNA samples must also be entirely...

...immunoprecipitation applications, but none of the traditional tags were suitable for both applications. To prove...from non-vertebrate systems. Preliminary testing indicated that inntags IT5, IT6, and IT10 expressed well...and the chaperone Ydj1, both of which display sophisticated interactions with multiple other proteins. Again...detrimental effects on cell size and growth rate, indicating that they compromised protein-protein interactions...These tags are easy to use in multiple common applications. Due to their limited effects on native protein...particularly in single-molecule or high-throughput applications. Perhaps IT5 and IT6 will even replace our older...

...Balancing research with medical practice as an MD/PhD. Darrel does 20% clinical work 80% research and was...for Regenerative Medicine at Boston University Medical School. Darrell’s research focuses on the lungs...Center for Regenerative Medicine, and within the biomedical research community. Along these lines, Darrell...Pathology and Laboratory Medicine. He attends in the Medical Intensive Care Unit, the Pulmonary Consultation...the Pulmonary outpatient clinic, all at Boston Medical Center. In addition he conducts basic stem cell...Regenerative Medicine of Boston University and Boston Medical Center. Special thanks to our sound engineer/producer...

...conventional microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I... science communication and outreach - if you'd like to learn more about Science communication check out...listening to talks all day. But nope, not at the American Society for Microbiology 2018 Microbe meeting....biomolecule-based dyes. This technology is widely applicable as bacterial expression of dyes allows dye visualization...Bacterial Genome Engineering Learn about Science Communication using Twitter  Resources on Addgene.org Check...

...provided here for reference: OpenWetWare Preparing chemically competent cells (protocol)  Preparing electrocompetent...protocol)  Untergasser Lab Preparation of chemically competent cells (protocol) Preparation of electrocompetent...until we are ready to perform transformations. A typical 200mL bacterial culture produces 20mL of competent...transformation protocols below: OpenWetWare Transforming chemically competent cells (protocol)  Electroporation (...protocol)  Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of...being performed at Addgene, this adds up to a significant time savings. You can also save yourself time...plasmid is introduced to bacteria, the bacteria is typically grown in liquid culture without antibiotics to...

...be part of the answer. Problems with written publications Online tools like ResearchGate, GoogleScholar...from the science of others. Improving science communication through video     Video abstracts about research... to be that video is the future in terms of communicating ideas. In 2012, for example, 3 billion hours...video usurps the written word as the means for communicating research. Short videos of 3 to 5 minutes provide... researchers to leverage the video medium to communicate their research more effectively.” How to share...concentration of citations, 1900–2007." Journal of the American Society for Information Science and Technology...study." Journal of Librarianship and Scholarly Communication 2.2 (2014): 2. Additional Resources on the Addgene...