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...anneal with each other, while the 5’ flaps containing the original DNA sequence anneal with each other. Excision...identify dozens or hundreds of particles surrounding each of the highly-visible beads on the cryo-EM grid.... samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM samples can be damaged at the air-water...plunge freezing represents a convenient, effective approach to protecting your cryo-EM samples from damage...

...expression, and viability. Gates are the parameters the machine uses to differentiate between variations in those...tumor cells, immune cells, blood cells and more), each coming with a different size and structural complexity...A) A flow plot showing two stains in relation to each other. Using a quadrant gate, I can divide my cell...cell population into DN, DP and SP (for each stain respectively). (B) Instead of a quadrant gate, I am...occur when a cell is pulsed by the laser. Within each type of scattering, you also discovered that there...

...obvious that a desired career outcome is not easily achieved. Of note, it was highlighted during the conference...Positive Points for Trainees - Taking Steps to Achieve a Successful Career Career Advice from the Career...showing researchers that they can take steps to achieve success in their careers. The take-away message...can take the time to do this, you should always reach out to your peers and other mentors who can help... postdocs, or professors isn’t nearly enough to reach diversity goals. Universities need to show that ...

...I have a list of what I really need in order to reach my research goals. Would I be able to get access...lack of resources, then ask for suggestions to meet each others’ needs and limitations. Everything is negotiable...research training to help you find other solutions to reach your goals. For example, a junior investigator turned...seem, you should consider it if it will help you achieve your research goals, even if that means saying ...a laboratory research management consultant and coach in Boston, MA. He specializes in assisting research...

...happening everyday, it’s not possible for us to reach everyone. Therefore, to make our library more useful...the repository? Let us know! You can notify us to reach out to potential depositors in two ways: 1. Fill...include any additional details that will help us reach out to the corresponding potential depositors. ...plasmid. While we cannot guarantee that everyone we reach out to will end up depositing, we hope this campaign...

...histone modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs...Both of these methods also recruit epigenetic machinery - but is there an advantage to using direct epigenetic...Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based...Safi A, Song L, Hilton IB, Crawford GE, Reddy TE, Gersbach CA (2017) CRISPR–Cas9 epigenome editing enables...Ruan K, Wang F, Xu G, Hu R (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discovery...

...our bloggers each and every month, we've decided to start an "Editor's Choice" series. Each month, I'll...was a fantastic month for the Addgene blog. We achieved roughly 50,000 views over the entire blog and ... Expression in Mammals Recombinase-based State Machines Enable Order-dependent Logic in vivo As always...

...related LCR technique uses two sets of probes to achieve exponential amplification. What does fidelity mean...substrates. Thus, high fidelity ligation can be achieved through a combination of the intrinsic selectivity...four upstream probes and four down-stream probes, each differing only in the base at the ligation junction...represented. Sixteen separate pools were prepared, each with a different complement strand covering all ...the template. The probes were designed such that each possible pairing resulted in a product of a different...

... used in limited circumstances. An alternative approach is to flank the desired change with 4-5 silent...from mini-λ or 4) from the lambda red phage itself (each of these will be discussed in more detail below)...can be modified with any of the below described approaches.    Bacterial strain with integrated defective...requires drug selection.  One drawback to this approach is that it requires the generation of bacteriophage...modification into a different background, if needed. This approach is best suited for modifying plasmids or BACs ...

... on genome sequencing. Used in concert, these approaches can provide the researcher with a reasonable ...(Fu et al. 2013; Pattanayak et al. 2013). This approach misses larger mismatches (e.g. six nucleotides... may be to integrate both biased and unbiased approaches. As suggested in a review by Tycko et al., 2016...a highly accurate manner. Using both of these approaches may not be necessary for every CRISPR experiment...Mark J., et al. "Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases....

...found yourself staring at a browser full of tabs, each open to a different antibody option. Or you may ... options in the Antibody Data Hub. Data reports  Each antibody submission is used to create a data report...Curating the data reports Just like our AAV Data Hub, each data submission we receive is checked by Addgene's... Rachel Leeson...

...a plasmid or viral origin. The type of cloning approach you choose ultimately will influence the quantity... for the extraction. Restriction enzyme based approaches can require several hundreds of nanograms or ...assembly or Golden gate assembly use a more direct approach, allowing scientists to clone from a smaller amount...will likely require micrograms of plasmid DNA for each reaction. As a consequence, the preparations for...

...different enough in size to be easily resolved from each other. However, by choosing an enzyme or enzymes...others. When choosing restriction enzymes for this approach, it is often a good idea to choose two different...orientation so long as the expected products from each orientation are different sizes. Simply run the ...

...Addgene Careers series, we sit down with Addgene Outreach Scientist Jessica Welch to discuss her career ...scientific community. Jane Hannon Our Marketing & Outreach Specialist Jane Hannon discusses her career path...scientists looking to expand their international reach. Joel McDade Joel McDade, Addgene's Business Development...

...separate different components in a liquid sample. It achieves this by using centrifugal force, which is generated...keep in mind when using any centrifuge. However, each centrifuge may have specific instructions so be ... pellets form in approximately the same place in each sample. Place the centrifuge’s internal lid on the...

...light used to activate the first PA-FP does not photobleach the second, and vice versa. Finally, as every...contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce... PA-FPs is photoactivated, imaged, and then photobleached. Additional PA-FP molecules are then activated...still do not emit the large number of photons achievable with photoswitchable dyes. Correlative light ...297.5588 (2002): 1873-1877. PubMed PMID:12228718. 2.Subach, Fedor V., et al. "Photoactivatable mCherry for...

...facts. Because, similar to scientists, the legacy of each government and generation is in the veracity of ...the lab, she dabbles in science policy, public outreach, and restorative justice.  Follow her on twitter...where these issues hit hardest. According to the Guttmacher Institute, most women of reproductive age live...galilei-dialogues-concerning-two-new-sciences). This is the approach policy makers take in abusing climate science ...tirelessly to get the publications, grant funding, and teaching experience that it takes to complete your resume...

...consensus lentiviral expression vector and attached a DNA barcode to each to make the library usable for pooled...proliferation, survival, differentiation, and apoptosis. For each of these 17 pathways, they identified a set of one...researchers didn’t have to discover new ways to activate each of these pathways. Rather, they relied on published...

...method that uses iterative staining and chemical bleaching to allow for high-resolution imaging without degrading...IBEX: An iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues...). IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells... Rachel Leeson...

...2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their roles. Lobe Domain...crRNA:tracrRNA duplex — Cas9’s two lobes move toward each other into the “closed” conformation shown in Figure...test DNA bases that are already snugly paired to each other? At this point, the protein can switch between...guide RNA (Cofsky et al., 2022; Osuka et al., 2018). Each matching DNA:RNA pair gives just enough stability...own advantages—and blind spots—to these questions. Each new structural insight also spurs the next generation...