...might think that being able to figure out what went wrong with a PCR is a pretty niche skill; outside of a...about seven different skills to you, you are not wrong. Problem-solving overlaps heavily with other skills...core part of our academic training.  Developing strong problem solving skills is just a matter of applying...As such, scientists are uniquely prepared to be strong problem solvers both in and out of the lab. The...position. Of course, there are limits to how far strong problem solving skills alone can take you - just...effectively does not mean I am qualified to be an astronaut. But, knowing that this skillset is a part of ... Ashley Waldron...

...be used for sparse labeling of neurons for tracing experiments.  Neuronal tracing experiments: While synthetic...broad number of cell types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than ...that produce sparse labeling would be useful for neuronal tracing experiments where lower levels of labeling...labeling makes it easier to trace a neuron’s projections. Generating animal models: Transgenic animals allow...for retinal gene therapy Find more about precise neuronal targeting using AAV technologies Browse all of... H.P., Krol, J., & Roska, B. (2019). Targeting neuronal and glial cell types with synthetic promoter AAVs...

...Nature’s toolbox. Natural plasmids, also called environmental plasmids, are all around us! A plasmid is a ...through transformation (cellular uptake from the environment), transduction (viral infection by bacteriophages...their host organisms to thrive in competitive environments. The genes encoding these functions form the...less aggressive ways to survive in competitive environments — like by living where no one else wants to....others can’t. This ability to endure a harsh environment is distinct from the benefit of resistance plasmids...lightning-mediated gene transfer in soil. Applied and Environmental Microbiology, 67(8), 3440–3444. https://doi.... environmental plasmids...

... photons are absorbed by matter, they promote electrons within atoms to a higher energy state. This process... called excitation. After a short period, the electrons revert to a lower energy state, whereby a photon...those of absorbed photons. When photons excite electrons of a fluorophore, they can do so at a range of...by the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few...absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure 2: Excitation... real-life advantage of a brighter fluorophore? Strong brightness makes it easier to distinguish a signal...and FITC an organic molecule), their bleed is so strong that these spectra almost overlap. Hence, they ...

...bind to coronavirus spike proteins (Wrapp et al., 2020), which sit on the surface of coronaviruses and enable...injected the spike protein from two different coronaviruses into a llama over several days and isolated ...sybodies that hypothetically contain ones that bind strongly to your target protein. The Seeger lab designed...enrichment step for identifying sybodies that bind strongly and specifically to the protein of interest. The...Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell ... carriers collection Find other COVID-19 and Coronavirus Plasmids & Resources ...

... stem cell-derived neurons expressing ASAP5-Kv. C) Cultured rat hippocampal neuron expressing pan-membrane...synaptic events. Neuron, 112(22), 3680–3696.e8. doi: https://doi.org/10.1016/j.neuron.2024.08.019.  ...ePEmax3 optimized for dicots. NC: nucleocapsid RNA chaperone, RT: reverse transcriptase. Right: T1 tomato plants... gene to confer resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after...antibodies for neurodegeneration research   By Ashley Waldron We recently added two antibodies against important...

...only a few, very strong associations.   Unexpected results Their results showed 96 strongly correlated genetic...accessible to the research community. Sean Misek and Aaron Fultineer in the Beroukhim and Boehm labs pulled...either European or East Asian ancestry, Sean and Aaron knew that their study had significant statistical...mapped to a SNP would be ignored. Working with Lia Petronio and Katherine Huang from Broad’s Pattern team,...varying burden across ancestries. Sean A Misek, Aaron Fultineer, Jeremie Kalfon, Javad Noorbakhsh, Isabella...Isabella Boyle, Joshua Dempster, Lia Petronio, Katherine Huang, Alham Saadat, Thomas Green, Adam Brown,...

...copy. This is a versatile technique, and is less prone to off-target mutations. Ultimately, successful ...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is also a... evolved specifically for toxicity would allow stronger counter-selection than one merely co-opted toward...rhamnose regulon, we inserted a copy of rhaS with a strong synthetic ribosome binding site (using the online...isothermal assembly master mix. Introduction of the strong transcriptional terminator BBa_B1002 (from IGEM...Modification of Proteobacterial Genomes." Applied and environmental microbiology (2019): AEM-00990. PubMed PMID:...chromosomal gene replacement in Escherichia coli." Appl. Environ. Microbiol. 81.15 (2015): 5103-5114. PubMed PMID...

...extremely strong affinity for one another. Actually the bond between them is one of the strongest noncovalent...that scientists began to exploit biotin and its strong interaction with avidin for molecular biology and...are useful for biotinylation in different microenvironments. There are also reversible and cleavable biotinylation...attached to a photoreactive arylazide group. Under strong visible light, the arylazide group becomes reactive...Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology of Biotin Attachment ... PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic biotinylation of proteins...

...neuroscience by granting targeted control over neuronal activity. While they serve similar purposes, both...scale of milliseconds, and can precisely link a neuron's activation to behaviors. Scientists continue to... mutated to have faster off kinetics and allow neurons to repolarize faster.  Chemogenetics does not offer...Administering the drug once can result in several hours of neuronal excitation or inhibition. This sustained activation...tissues, cell types or even subcellular regions of a neuron. Optogenetic approaches excel at discrete cell ...precise spatiotemporal control of a small subset of neurons is necessary, use optogenetics. If broad control...

...monitoring the activity and biochemistry of living neurons in an effort to understand the connection between...available beam splitters. In addition to imaging neurons, Griesbeck adds that this color change has a particular...refined by a secondary screen in rat hippocampal neuron cultures. The optimized Twitch sensors allowed ...researchers to see tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes...transition metal ions, such as zinc, copper, and iron. “These are essential metal ions, but they are also...calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014 Jan 5. doi...

...sterilization as mycoplasma can even escape 0.2-micron filters. Figure 1: Contamination! Tissue culture...shortly. Once they are present in the laboratory environment, they are difficult to eradicate. You can suppress...maintenance of the cell lines. Providing such an environment for the lab facility will not only reduce mycoplasmas... you are careful enough to provide a sterile environment for work, it will soon become a habit before ...separate footwear for the lab to avoid bringing in environmental contaminants.  Figure 2: An Addgenie...lines for assay or analytical purposes. Use 0.1-micron filters as are they are a better way of avoiding...avoiding mycoplasma, rather than using 0.2-micron filters, for media filtration. There is no 100% assurance...

...reversibly degrade proteins of interest fused to a degron, a domain that induces degradation. In plants, ...ligase complex, promoting its interaction with the degron sequence and ultimately its degradation. While ... needed for AID. By tagging proteins with a 7kD degron called mAID and expressing the essential plant ...channels that can be used to silence the activity of neurons. Earlier this year, the Hegemann lab and collaborators... cysteine.  With the introduction of MerMAIDs, neurons can be turned off quickly and with high precision... a valuable optogenetics feature when studying neuronal activity. The researchers identified MerMAIDS ...

...there was strong expression of Cpf1 and GFP in the targeted brain region and ~75% of neurons were co-transduced...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...multiplex edit 3-4 genes in a primary culture of mouse neurons and in vivo. For these experiments, cells were ...-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted loci.  See the chart...

...Murphy’s law definitely applies: what can go wrong, will go wrong. The trash removal service slams dumpsters...useful transferable skill. Being able to stand up in front of an audience and convey your ideas clearly and...project. So whenever you freeze, or when things go wrong, ask yourself, “What next step will help me achieve...term “diving board” so you end up acting it out in front of a very confused group of visiting Chinese undergrads...incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck gets uploaded. Your mic...

...Tet-on transgene vector. Degron tags Tagging relevant proteins with a degron tag can provide inducible...removed. Degron tag systems include small molecule assisted shut off (SMASh), auxin-inducible degron (AID)...SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron tags,...

... RNA-sequencing to identify neuronal class and deep sequencing of neurons from each class to fully characterize...Addgene.  C. elegans open resources The C. elegans Neuronal Gene Expression Map & Network (CeNGEN) The CeNGEN...comprehensive gene expression atlas of every C.elegans  neuron. Scientists are currently doing this using two ...this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin dependent manner. This...Gene Expression Map of an Entire Nervous System." Neuron 99.3 (2018): 430-433. PubMed PMID: 30092212. PubMed...

...across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by action potentials ...perceived signals to physical actions. Understanding neuronal pathways and the function of certain cells depends...be directed to specific types of cells, such as neuron subtypes, thanks to viral serotype specificity ...available VSDs has been successful even at the single neuron level in vitro. In vivo monitoring has also been...membrane as well. Another flavor of these tools is Voltron, which uses rhodopsin as the voltage sensing component...voltage is particularly physiologically relevant in neurons, myocytes, and endocrine cells. It follows that...

...Chemical Synapses in the Brain. Neuron 68:843–856. https://doi.org/10.1016/j.neuron.2010.11.021  Gambarotto D...techniques like super-resolution microscopy or electron microscopy. Since its introduction in 2015 by ...2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment...helped researchers gain a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux... Ashley Waldron...

...norepinephrine (NE) expressing neurons and GFP (green) to label neurons transduced by each serotype. ...challenge to scientists. What works well in one cell or neuron type may not work well for a neighboring cell type...express ChR2(H134R) in A2 norepinephrine expressing neurons. We tested four serotypes containing an expression...84%) were both excellent at infecting our target neurons (Figure 1). However, this one set of experiments...Perspectives on Data Sharing in Deep Brain Stimulation. Frontiers in Human Neuroscience, 14(December), 1–12. https...

...biological studies, including investigations into environmental pollutants and health, embryo growth, brain ...example, specific tissues such as blood cells or neurons can be fluorescently labeled, allowing researchers... for neuroscience investigations. For example, neuronal activity can be tracked in whole brains of swimming...high-resolution mapping of the brain by uniquely labeling neurons. Zebrafish are also amenable to manipulation of...consequences of activating or silencing specific neurons in living larvae. In addition, zebrafish have been...PubMed PMID: 18845710. Kim, Dal Hyung, et al. "Pan-neuronal calcium imaging with cellular resolution in freely...

...make them ideal for studying in a laboratory environment. We’ve covered five model organisms in a previous...candidate for introductory teaching and research environments (Idalia & Bernardo, 2017).  Escherichia... disease-relevant genes in such a controlled environment displays how essential the frog is as a biomedical...genome, and is easily maintained in a laboratory environment. What sets Arabidopsis apart from other plant...Regeneration and Aging Research: A Mini-Review. Gerontology, 57(6), 565–571. https://doi.org/10.1159/000323761...the Axolotl Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294...

...Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021 Walters..., 2023). It may be a good choice for targeting neurons, with high transduction frequencies in the central...to target the central nervous system, including neurons, AAV5 is generally the best choice for targeting... is derived from AAV2 and engineered to infect neurons and undergo retrograde transport, traveling up ...

...molecule at all, leading to inconclusive results, erroneous conclusions, and wasted time. In fact, many have...recommendations for improving antibody use and reliability (Roncador, et al., 2015; Taussig, et al., 2018; Uhlen, et...that antibody works as expected, you will see a strong antibody signal in the retinal sample, but little... your antibody (B). Your results show a lovely, strong band from the cells transfected with your protein...Nature 518:27–9. https://doi.org/10.1038/518027a  Roncador G, Engel P, Maestre L, Anderson AP, Cordell JL... Ashley Waldron...

...mouse neurons. D) Membrane voltage traces of action potentials in a HcKCR1-expressing mouse neuron without...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts to engineer...of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies with ...antibody anti-c-Myc [9E10] now available! by: Ashley Waldron The majority of Addgene's more than 50 antibodies...

...at Addgene, I get to see everything that can go wrong with a plasmid. One of the most frequent problems...smaller plasmids are formed. Viral vectors are prone to recombination In lentiviral vectors, recombination... elements. Knowing how to amplify recombination-prone plasmids and testing your prep can save you the ...H (2012) Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3...Genetic recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular...

... Frank Graham showed HEKs display many immature neuron-like qualities, which is inconsistent with kidney...is next to the kidney and is known to have some neuronal qualities.) While HEKs probably aren’t a great...researchers using the cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible..., et al., Preferential transformation of human neuronal cells by human adenoviruses and the origin of ...Produce Recombinant Proteins and Viral Vectors. Front Bioeng Biotechnol. (2021) (9):796991. DOI: 10.3389...

...equipment always helps. But how does one build a strong collaborative research team motivated by similar...your lab team and foster an effective working environment? Tom Ellis was offered a start-up package that...ways to improve the lab. “An effective working environment for research is totally dependent on communication...while others are more productive in a handsoff environment. “Treating people fairly does not mean equally...selectively picking the first couple of hires to build a strong, collaborative team. And take advantage of the ...

...error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error prone PCR,...bacterial growth conditions. Expression of an error prone polymerase I (PolI3M) is capable of mutagenesis ...PCR, EvolvR uses an error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m...   Figure 1: EvolvR consists of an error-prone polymerase (PolI) that is fused to a nicking Cas9...

...plasmid for expression in forebrain GABA-ergic interneurons (83895). See our Serotype Testing AAV inventory...with ever more efficient tools for light mediated neuronal control. The advantages of optogenetics lie in...system that allows for specific manipulation of neurons. We have expanded our AAV collection to include...targets expression of ChR2 to forebrain GAB-ergic interneurons through the mDlx enhancer element. Plasmid...DREADD acts through the Gq pathway and promotes neuronal firing. Plasmid Serotype Name Depositor 44361...

...signaling pathways in specific cell types (typically neurons). While all of these viruses are popular, pAAV-...pAAV-hSyn-DIO-hM4D(Gi)-mCherry can be used to induce neuronal silencing in vitro and in vivo (Krashes et al..... The human synapsin promoter further specifies neuron-specific expression of the DREADD (Kügler et al...Lowell BB. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J Clin Invest..... Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an...

...skills in strong relationship-building and influence. You’ll need a large deal of influence and strong relationship-building...software product management was either. Coming from a strong background of undergraduate research straight to...energized from interacting with others, and have strong communication skills to tailor your message to ...implementation review. The developers discuss which front-end and back-end coding frameworks to use. I mostly...

...29] Photoreceptors [30] Otic hair cells [31] Neurons [32, 33, 34] You can find a variety of plasmids...have been successfully transdifferentiated into neurons and β-cells [40-42]. Reprogramming and transdifferentiation...S.C. Zhang, Directed differentiation of dopamine neurons from human pluripotent stem cells. Methods Mol ...human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc, 2012. 7(10): p...pluripotent stem cells to mature functional Purkinje neurons. Sci Rep, 2015. 5: p. 9232. PubMed PMID: 25782665...Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 2010. 463(7284): p....

...increased iron serum levels and early-onset cataracts, is characterized by point mutations in the Iron Response....  In as little was one minute BASU generated a strong streptavidin signal (~250-fold enrichment) when...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, ... loss of binding correlated with increased serum iron levels seen in HHCS patients. These are just three...

...somewhat similar to an academic research environment” with a strong focus on publishing, but still made it...initial resistant populations can be eradicated up-front quickly. This approach is in contrast to the current...at cancer cell resistance to ABL1 inhibitors in chronic myeloid leukemia. Bhang says the work highlighted...2015): 440-448. Pubmed PMID: 25849130. 2. Hata, Aaron N., et al. "Tumor cells can follow distinct evolutionary...

...9514755. Richard Gronostajski: NFI-related diseases of neural development Richard Gronostajski is a professor...Paula Videira, Michael McMurray, and Richard Gronostajski who each are being awarded free plasmids form...they affect brain and muscle development. The Gronostajski lab has generated mice wherein it's possible...Campus in Aurora, Colorado, USA.     Richard Gronostajski is a professor at the University of Buffalo....

...could ever do! However, there is quite a bit of upfront research you have to do to find said robot and ...into the robotics spirit include: Start using electronic multichannel and single channel pipettes. These...help keep things more organized. Cap tubes with a micronic cap sealer. This is a simple table top device ...has a roller to go over caps and push them into micronic tubes instead of pushing them in one by one. It... It seems very simplistic and it isn’t electronic but it is one of the most helpful machines I have used...

... an extensive infrastructure of molecular and electronic companion technologies that has transformed the...CRISPR/Cas9-mediated genotyping, as well as our electronic reader that provides portable quantification ...Leslie Dan Faculty of Pharmacy at the University of Toronto. His research is focused on developing cell-free...biology applications for human health and the environment.     Alexander A. Green is an Assistant professor...4. Messina, Jane P., et al. "Mapping global environmental suitability for Zika virus." Elife 5 (2016):...

...conserved in mammals, expressed in neurons, and are especially abundant in neuronal synapses. shRNA silencing ...way to guide inheritance that might be less error prone than DNA replication. He too mentioned how “surprising...regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University then spoke about using...treatment resulting in major problems in hospitals. Dr. Qimron proposes using bacterial phage vectors with CRISPR...

...antibody is produced from a new set of animals), and wrong application (different experiments/conditions can...that this antibody has specific binding and has a strong signal. We call the unique pattern of the antibody...7s) then the signal is weak and vice versa for a strong signal. However, exposure time and its relationship...compare exposure times of our samples. A prominent, strong signal has a shorter exposure time than the negative... neuroanatomy without the fear of referencing a wrong section. The antibody screening and development ...