...dilute it, err on the side of caution when adding buffer back to the solution. Add a bit less buffer than...concentration to avoid over-diluting, and make sure to remeasure your titer after diluting to confirm. Luke...allow you to add many more viral particles to your solution while still using a volume that’s compatible with...surface of the filter membrane with the surrounding solution to ensure excess viral particles do not remain...remain adhered to the membrane. Be careful when diluting - if you end up concentrating your AAV above your...

...implement solutions for the stakeholders. Finally, software product managers test the solutions with the...cycle over again to refine the solution, or to come up with a new solution.  Now, you may be wondering how...the time, the problem to solve is clear, but the solution is more complicated. You’ll need to be creative...able to work with others to come up with a great solution.  How to get into software product management?...customer help tickets, and respond when I have solutions. We use these to help us know what to work on ...

...Nikolai Braun and Keira Havens, co-founders of Revolution Bioengineering. Read their previous blog post...the public in an open, responsible manner. At Revolution Bio, we are addressing this challenge directly...people can decide for themselves if they want this beautiful flower in their front yard or not. By developing...investigated on limited funding and time. While institutions that focus on creative problem solving like ... science, these tools sometimes have limited distribution. We hope that will change with Addgene’s expansion...project. We’ve shared some of the concerns that Revolution Bio had to address as we developed the color-... in the United States Air Force. She left active duty to pursue a degree in a synthetic biology laboratory...

...description of the RFA and funding institution. Emphasizing how your solution fits into the mission of the ...funding from a variety of government and academic institutions. New graduate students may assume that their...advisor may choose to allot some if not all grant duties to trusted students. For example, if the proposal...the mission of the application and the funding institution. Moreover, avoidance of ‘to be’ verbs creates... of the statement, use it to confirm that your solution is the best approach to the research challenge...the intended protocol, and offers alternative solutions to demonstrate thorough thought planning on the...

...These events have also started progress towards solutions. The 2015 Boston FoR meeting was designed to explore...further data is necessary to determine the best solutions. The event also culminated in a joint American...students and postdocs broadly – and thus PI’s and institutions in turn. The first is the passing and upcoming...the NIH recommended pay scale – used by many institutions to set postdoc wages. The second event of note...simply being able to categorize all postdocs at institutions, will be challenging. At the same time, there...level, FoR is currently compiling a database of institutional responses to the FLSA ruling. Similarly, from... and graduate students, and started to define solutions but this year, together with Academics for the...

...expression following transduction with serial dilutions of a viral prep. This method is particularly useful...Fluoresence Titering Protocol Another popular approach utilizes the antibiotic resistance genes carried on transfer...plasmids. Target cells are transduced with serial dilutions of a viral prep, treated with the antibiotic, ...intensive. Target cells are transduced with serial dilutions of a viral prep, genomic DNA isolated, and primers...months but should be considered in labs that will routinely be producing virus or require precise titers. ...their benefits and drawbacks. Labs that do not routinely produce virus may want to choose more straightforward...colony-forming unit assays, whereas labs that routinely produce virus may want to consider the more accurate...

... their PhD and postdoctoral training. Addgene executive director Joanne Kamens has written a 5-part blog...pursue leadership opportunities at the local, institutional, regional, and national levels. These skills... But how does one gain such experience? Your institution may offer occasional workshops on leadership ...Harvard Medical School and then Boston University (institutions where I’ve worked as a postdoc). I soon realized...that many of the issues faced by postdocs and institutions are not local but are national issues. I applied...many changes to the  Board of Directors and the Executive Director changed twice. I had to learn how to ...to the Board of Directors and liaise with the Executive Director. As members changed, I had to learn how...

...that is shared across all CRISPR types; the fast evolution of these adaptive bacterial and archaeal immune... in archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...., & Koonin, E. V. (2014). Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Research... E. V., & Makarova, K. S. (2019). Origins and evolution of CRISPR-Cas systems. Philosophical Transactions...Zhang, F. (2017). Diversity, classification and evolution of CRISPR-Cas systems. Current Opinion in Microbiology... J., . . . Koonin, E. V. (2015b). An updated evolutionary classification of CRISPR–Cas systems. Nature... Yakunin, A. F., . . . Koonin, E. V. (2019). Evolutionary classification of CRISPR–Cas systems: a burst...

...spectrum and emission spectrum. Fluorescent dyes utilize these fluorophores by combining them with a molecular...These variations directly impact the way you can utilize these dyes. The most-used fluorescent dye is fluorescein... between multiple dyes during analysis. Image resolution is impacted by several factors. Firstly, brighter...have been specifically designed for use in super-resolution microscopy techniques, such as Stimulated Emission...These dyes have specific properties that enable resolution beyond the diffraction limit of light. Dyes like...properties that position themselves for super-resolution usages, such as superior brightness, photostability...brighter and more stable fluorescence for super-resolution applications. Other current advancements include...

... sticking pipettes into solution bottles directly, rather decant the solutions into disposable sterile... up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine. Twice...that you also have the power to take the added precautions necessary to keep your cells from becoming contaminated...things more manageable, but it also keeps stock solutions from getting contaminated. Make sure cells don't...So if you, for example, split cells on a Monday diluting them 1:10, you should be able to split on the ...

... positives [2, 4]. Solutions to the polymer problem There are some simple solutions available for researchers...GFP to study the protein's behavior in cells, in solution or something in between, you are using a tag with...ruin your day (and experiment) are listed below. Solutions to avoid these all too common issues follow. ...with all of these problems. The simplest and best solution is to use truly monomeric FPs. EGFP and other ...they are designated mEGFP, mVenus, etc. A word of caution is needed because FPs not derived from GFP, i.e...depend on protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing...

...which can reduce the potential image resolution of super-resolution fluorescence microscopy. Their size...about 2 nm, which can lead to higher resolution for super-resolution microscopy. Nanobodies can be labeled...animal sacrifice Poor tissue penetration Less resolution due to larger label displacement Require separate...penetration Can be labeled with multiple dyes Greater resolution due to lower label displacement 2° incubation... Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein...

...vector solution, and generally these methods measure different characteristics of the solution. These ...can also be altered by freeze-thaw of the viral solution (Lock et al., 2010). Infectious titer: the ...quantified by electron microscopy of a viral vector solution. Since this technique is intensive and requires...requires an electron microscope, it is not routinely performed on all new viral vector preparations. Addgene...facilities report the physical titer of a viral solution (Figure 2). Because physical titers are used for...starting template is the purified viral vector solution (which contains viral capsid proteins) as opposed...

...Blainey, P.C. (2024). CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled...valuable tool for microglia research and broader therapeutic applications.  Find microglia-targeting AAV plasmids...https://doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System...has developed REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large...Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector... The authors used REVeRT to functionally reconstituting coding sequences for a range of applications...mechanisms of delivery and the potential for therapeutic success. Find REVeRT plasmids here! Riedmayr...

...the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than studies ...or cell culture. For example, Scott Lowe’s lab utilized mice to study how different mutations in leukemia...important human disease model with potential for therapeutic drug discovery as it is estimated that 75% of...they can also be used to easily screen multiple therapeutic compounds in one experiment. Zebrafish (Danio...to name a few. For example, Andrew Leifer’s lab utilized the worm to study calcium signaling in the entire...al. "Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans." Proceedings...Drosophila melanogaster and the role of the fly in therapeutic drug discovery." Pharmacological reviews 63.2...

...Structural problems in academic research and potential solutions During the first couple of sessions of the symposium...his or her chosen career path,” but individual institutions can have many different names for the positions...From Panel Session 2: Defining the Postdoc: An Institutional Perspective. This lack of complete information...workforce, and we are beginning to identify potential solutions. Keynote speaker Paula Stephan did a fantastic... articulating many of these problems and some solutions. The structural problems seem to boil down to ...here). Importantly, the panel offered some great solutions to these problems and some that stuck out to me...those at the FOR symposium and will enact their solutions as soon as they become actionable. Additional...

...cytosine-to-thymine substitutions in target sequences (Banno et al., 2018). These substitutions were limited ...the utility of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively... is done with recombineering, a technique that utilizes phage recombination machinery to promote homologous...especially for larger modifications. What’s the solution to this inefficiency? Use CRISPR to make it a ...introduce point mutations in P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae...respectively. Substitutions seem most efficient when using a nickase version of Cas9, but were also successful...

...common solutions to avoid background staining when doing species on species IHC. (A) One solution is to...fluorophore) directly attached to it. (B) Another solution is to combine your primary and secondary antibodies...aren’t free to bind endogenous IgGs. (C) A third solution is to use Fab fragments against the sample species...still see non-specific staining! Hopefully, these solutions will help get your experiments back on track.  ... or immunocytochemistry (ICC). Not all of the solutions described here may be as effective for other techniques...

... super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging...which reacts with O2-benzylcytosine(BC) ligands (Gautier et al., 2008). Although based on the same original...multiple proteins but label each separately before reconstituting, choosing your colors depending on the needs... want to use far-red rhodamine dyes for super-resolution imaging, HaloTag gives better performance than... (2019). Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and...7), 547–556. https://doi.org/10.1021/cb900062k Gautier, A., Juillerat, A., Heinis, C., Corrêa, I. R., ...

...The most commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as ...the system. Since then, multiple biotech and pharmaceutical companies (along with some research laboratories...It’s also a methylotropic yeast, meaning it can utilize methanol as its sole source of carbon and energy...distantly related to S. pombe and S. cerevisiae; evolutionarily it evolved much slower and bears more similarity...the expression of recombinant proteins by the pharmaceutical and biotech industries as a protein expression...greatest hurdle for genome engineering. A unique evolutionary feature of K. phaffii, relative to S. cerevisiae...