...rapid multi-part DNA assembly method - read on to learn how to use it in your research. The starting point...cloning used short sticky ends, LIC employed the exonuclease activity of T4 DNA polymerase to create longer...be present in the reaction mix, limiting the exonuclease activity to the first occurrence of that nucleotide... intermediates” through PCR and imprecise T4 exonuclease activity, overcoming the requirement for carefully...step, the addition of a single dNTP stops the exonuclease reaction. The PCR fragment and plasmid are then... DNA polymerase, Gibson assembly requires T5 exonuclease in combination with Phusion polymerase and DNA...

...back: David Gordon, Jeff Guo, Fengbo Zhou, Paddy O’Leary (UCSF). Our approach is to discover key human...FDA-approved). This is a big job, but under the leadership of Nevan Krogan at UCSF we’ve brought together...preclinical studies already. We are following up on these leads as we speak, with collaborators in Paris and New...affected in various ways by the pandemic, it is clear that we have to stick together (6 feet apart), to...shout out to Margaret Soucheray, Jeff Guo, Paddy O’Leary, Fengbo Zhou, Helene Foussard, and Dave Gordon)... plasmids for coronavirus or COVID-19 research, please email Addgene at [email protected] with your order...

...Avoid star activity. Some endonucleases (for example BamHI) are capable of cleaving sequences which are similar...cloned the plasmid yourself or obtained it from a colleague down the hall, it is always a good idea to take...incubating your DNA with restriction enzymes which cleave the DNA molecules at specific sites and 2) running...manufacturers make High Fidelity versions of the endonucleases and/or supply custom buffers as means to avoid...is not just necessary but also an easy process. Please vist Addgene's resource for plasmid verification...

...specific. Once inside the host cell, the virus unleashes its cargo – its own genome.   Fig. 1:...access to the host genome requires breakdown of the nuclear envelope, which occurs during mitosis. The most...and dividing cells. They accomplish this through nuclear localization signals which can traffic their viral...PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human immunodeficiency virus type 1 preintegration...PMC1368960. Additional Resources on the Addgene Blog Learn More about Lentivirus Listen to Connie Cepko Discuss...Resources on Addgene.org Browse Our Viral Vector Pages Learn About Viral Vector Biosafety Deposit Your Viral ...

... or profession, you need to make sure you have learned and mastered the fundamentals.   What are your ...of the office? Katz: I have a puppy and so I am learning how to train a dog with my daughter. It’s super...with our new dog, Daisy, or out on golf course! Learn more about Karen Katz, Esq Ms. Katz is the Director...Boards of BioBuilder and Brookline Interactive. Learn more about Carl Paratore, MBA, CPA Mr. Paratore ...Auditor at Tufts Health Plan. In that capacity he leads the Internal Audit function and is responsible for... MA. Additional resources on the Addgene blog Learn more about about Addgene in our "Inside Addgene"...

... see by this post’s chromatin illustrations from Leah Bury of Microscopic Art. I started thinking about... we definitely don’t have that you really want, please let us know! And if you study chromatin and want...indicated gene, and you can subscribe to these pages to learn when Addgene receives new plasmids containing these...t see the gene you’re looking for on this list, please consider searching directly on our website. ... 25. 95(5):625-36. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure...Addgene blog post about chromatin--multiple Addgene colleagues and guest bloggers have written posts describing...

...affected by light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success...can be affected by both autofluorescence and photobleaching. Researchers have long been interested in using...reality, Addgene depositor Takeharu Nagai and colleagues at Osaka University have developed Nano-lantern... cells. As Nano-lanterns are not subject to photobleaching/phototoxicity, they successfully imaged multiple...applications. For long-term imaging, especially where photobleaching and phototoxicity are a concern, these constructs...Addgene. Additional Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check...

...Blog Learn About our Cancer Colleciton Pages Use the ClonTracer Library for Lineage Tracing Learn About...to well-annotated cellular pathways). It wasn’t clear whether gene-agnostic bioassays could accurately...proud to have worked closely with our Addgene colleagues to make these available to the scientific community...10.1016/j.coisb.2017.02.002. We hope that by releasing these new and exciting plasmid collections (together...clinical sequencing studies. At the Broad, my colleague Cory Johannessen and his team have recently demonstrated...

...which cleaves the synaptic protein VAMP2, a protein essential for synaptic transmission. Cleavage of VAMP2...ever like to write about a recent plasmid deposit please sign up here.      Listen to this episode of the... it inactive, thus inhibiting neurotransmitter release. How is this light-mediated? The researchers split...split botulinum neurotoxin halves to enable VAMP2 cleavage, and suppresses neuronal transmission within minutes...differentiation. Furthermore, TALEN and ZFN require custom nucleases for each gene of interest. CRISPR-Cas9 tools have...

...polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific end cleaning enzymes (Aprataxin, Tdp2...Cas9 protein doesn’t immediately release from the broken end after cleavage, which may interfere with loading... the products of accurate repair are easily re-cleaved while indel products aren’t, so repeated cycles...latter products. As noted above, a single cycle of cleavage and accurate repair should take less than an hour...

...activate essentially any gene,” she says. The technical leap from mutating and deactivating a gene or genes to...published in Nature in December, Konermann and her colleagues in Feng Zhang’s lab describe just such a system...transcription activation processes. Konermann and her colleagues demonstrated the new system by screening for ...in those 15,977 human genes. Weissman and his colleagues demonstrated in a paper in Cell that they could...which guide RNA we use. What we have to do now is learn more about the rules.” He said that there is no ... These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to ...

...review see Maday et al., 2014). It is currently unclear which proteins facilitate transport of neuronal...expansion of RABV. This improvement resulted in a clearer picture of neuronal connectivity in the mammalian... mapping of the neuronal circuitry involved in learning and memory (Kohara et al., 2014; plasmids at Addgene...particle is taken up on one side of the cell and released again at another side of the cell after intracellular...transport without actually causing infection or leading to transgene expression within the first cell. ...development of more powerful AAV serotypes, and perhaps lead to controlled trans-synaptic spread of AAV vectors...21782903. McFarland, N.R., Lee, J.S., Hyman, B.T., and McLean, P.J. (2009). Comparison of transduction efficiency...

...that other technologies need to be discarded but clearly we are in an era of neuroscience research where... the technique is remarkably straightforward to learn and relatively cheap (compared to many other technologies...Program (NIH-NIDA IRP), with the stated goal of learning behavioral optogenetics in order to implement ... to share some of the tips and tricks and I’ve learned from numerous scientists at NIDA and some of the...region can result in a particular neurotransmitter release or post-synaptic current. Fortunately, if your ...variants [1, 2, 4]. And, of course, the best way to learn about the many different opsin variants and how ...University.  His interests include the role of learning and memory in opioid addiction and science communication...

... and ligands vary in their chemical structure, leading to differences in their excitation and emission...peak wavelength can also decrease the rate of photobleaching. Interaction with receptors: Understanding ...the choice of fluorescent dye. For instance, the cleavage of FITC-conjugated protein ligands is unaffected...experimental conditions. Common issues include photobleaching, non-specific binding, and quenching, which... reduce ‘muddying’ of the signal and facilitate clear differentiation between multiple dyes during analysis...states in response to a specific stimuli, which leaves only a small volume of fluorophores in an excited..., M., & Ghods, R. (n.d.). Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568...

...these molecules are clearly associated with cellular damage and dysfunction, it is unclear what the precise...Transposable elements such as long/short interspersed nuclear elements can generate dsRNA by bidirectional transcription...silenced, but dysregulation of these systems can lead to local dsRNA production. In the same vein, genotoxic...barring a loss of mitochondrial integrity that would release them into the cytosol. Fig. 2: Endogenous... of DNA damage, and these structures can in turn lead to genome instability. Thus, it’s important to strike...

... this cause and helped by expanding this team, leading monthly calls, and planning next steps. April Clyburne-Sherin...future of reproducibility workshops and lessons learned: Since early 2018 the team has grown to include...all levels of the scientific community. Lessons learned Instead of continuously talking about a problem...like to join or contribute to this collaboration, please check out the official website, follow the group...conversations on the history of the group, lessons learned, and to Sonali Roy, Magdalena Julkowska, and Jennifer...on Transferable Skills: External Collaborations Learn about the 2018 Minisymposium on Scientific Reproducibility...

...metabolism. Cancer cells promote oxidative stress leading to the degradation of one key enzyme (among others...built to protect us now seems to attack us by releasing mitogenic growth mediators and improving cancer... When it comes to breast cancer, Hoshinho and colleagues found that after injecting breast cancer cells...process is not fully understood, a team of researchers lead by Takayuki Kogure (Kogure et al., 2011) believes...Addgene blog Read our cancer related blog posts Learn about our cancer collection pages Read more about...resources page  Find plasmids for your research Learn about Addgene's viral service ...

... into close proximity with the target protein, leading to polyubiquitination of the target protein and...proteasome machinery through binding with cereblon, leading to rapid target degradation (Figure 1). Plasmids...ortho-AP1867) via various chemical linkers. Our current lead dTAG molecule is dTAG-13 due its immediate action...happy to share aliquots of dTAG-13 in the meantime, please address your requests for dTAG molecules and any...terminus is favorable for tagging. However, if it is unclear which terminus should be tagged, the dTAG lentiviral... interested in targeted protein degradation, and lead author on the recent paper describing the dTAG technology...

...s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more ...channelrhodopsin that’s successful in vivo. Using machine-learning to overcome channelrhodopsin design challenges... Arnold and Gradinaru Labs developed a machine-learning model to help them construct “designer” channelrhodopsins...and secure a fiber optic cannula to the skull, leaving the brain intact. When the fiber optic light was...Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables minimally... PHP.eB AAV capsid Read more about optogenetics Learn about the differences between chemogenetics and ...

...she made through this program lead to her current job. “One of my colleagues in the class was like, “Hey...people are,” says Susan. Her passion for insects lead her to start a PhD in entomology at the University...which Susan replied, “What’s a science writer?” Learning about SciComm by doing SciComm Writing had always...Evaluating evidence. Critical thinking. Being able to learn things quickly. Story telling. The ability to know...tips for using social media as a scientist here Learn more about storytelling in science ...