...happens, the solution can be cooled and the two phases mixed together. Lastly, the PEG solution needs to ... the iodixanol solutions and let me double-check that the iodixanol gradient solutions were prepared correctly...because it’s time consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate...to dissolve and requires monitoring so that the solution doesn’t get too hot because then the PEG will ...either by filtration, which takes time because the solution is viscous, or by autoclaving, which also takes...preparation. 15%, 25%, 40%, and 60% iodixanol solutions are carefully layered and then the viral suspension...titer for your AAV, remember to account for the dilution of the sample for the DNAseI digest. Use good ...

...temperature. If the agarose solution is too hot, the cells will die. If the solution is too cold, it will clump...out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time...survive at low densities - adding to the expense of dilution cloning. Taking a page from bacterial cloning...different densities for selection). A 2% agarose solution is then melted (and thereby sterilized) by microwaving...placed in the hood. Finally, a sterile 2X DMEM solution (diluted from 10X and containing both Hepes and... mm dishes only 1.5 ml of each are used). This solution is capped, swirled and tested for the correct ...the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body very sensitive to heat...

...repository has grown to include many CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative...cumulative numbers of CRISPR plasmid distribution and deposits (Figures 1A and 1B); the cumulative number...each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative CRISPR plasmids...Figure 2, the numbers on the X axis refer to distribution and deposits per year, rather than a cumulative...   Figure 2: Addgene CRISPR deposit and distribution by function and expression type. (A) Cumulative...4).    Figure 4: Addgene deposits and distribution by country. (A) Total CRISPR plasmids deposited...publication, “The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene...

...tasks, and then executing on them in a timely fashion while allowing for creative solutions to emerge. They... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...clinical research to discover and test the best new therapeutics. Consultants often work with such teams to help...associated with the business. Commercial teams routinely work with consulting firms on projects such as...future, and yet are incredibly difficult to find solutions to. There are many reasons why client situations...at the core of the suite of benefits a given therapeutic approach brings to patients, doctors, and insurance...others go on to work with commercial teams at pharmaceutical companies, nonprofit or global health organizations...

...DNA prep. You might see protocols that recommend eluting in water, Tris-EDTA (TE), just Tris buffer, or ... main factors that can affect DNA stability in solution are pH and DNase contamination.  Extreme pH can...activity. For these reasons choosing the correct solution is fundamental, and it will impact the time you...which experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water Figure...two of the choices that companies offer for the elution of plasmid DNA through their kit. The advantage... plasmid in every type of experiment. The two solutions may differ though in their capacity to maintain... to store it and for how long. The most common solution is to keep your plasmid at -20°C or even at -80...

...to the distribution of the submitted material under the terms deemed necessary by my institution or other... pre-publication plasmids" and set a desired distribution status. You can submit "Published plasmids":...description of the deposit. Select your desired distribution status: Hold for publication: we will initiate...pending QC: your plasmids become available for distribution as soon as they pass our standard QC process...that will make it easier for scientists to best utilize your plasmids (these can be protocols, GenBank ...information for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should ...Survival strain. Figure 10: Enter the growth and distribution information. Step 5: Verification On this page...

...c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer ...activation Optogenetics is a powerful tool that utilizes light to control and monitor individual living... both live cells and whole animals. The system utilizes the bulldozer-like quality of the ribosome during...red. This system achieves great specificity and resolution by using 6-12 phage PP7 coat protein binding ...create an Entry vector containing a promoter for constitutive (CMV, SV40, SFFV) or inducible (CMV-TO) cDNA...expression, sgRNA (U6) expression, shRNA/shRNA-miR30 constitutive (H1, U6, 7SK) or inducible shRNA-miR30 (CMV-...experimental purposes including (1) single or multiple, constitutive or inducible, gene knockdown with shRNA or shRNA-miR30...

...issues independently; for example, Harvard’s Therapeutics Graduate Program has a compulsory internship...It remains contested, however, whether a better solution would be to reduce the number of entrants into...in Boston - this time with the aim of finding solutions to some of the problems that were identified last...adequately supply these demands? Are there other solutions, such as increasing numbers in Master’s level ...the academic labour market. Her many notable contributions in this area include publishing the book ‘How...How Economics Shapes Science’ and contributing to the NIH’s Biomedical Research Workforce Working Group...2015 where we hope to begin developing concrete solutions to the problems looming over the academic research...

...2020). Directed evolution of adenine base editors with increased activity and therapeutic application. Nature...efficiency (Koblan et al., 2018). Later directed evolution efforts yielded an editor with more flexible sequence...DNA adenine deaminases. The team used directed evolution to create one from the RNA adenine deaminase TadA... TadA.   After seven rounds of molecular evolution, they obtained four adenine base editors (ABEs). ABE7.10...In this first, the Liu lab used phage-assisted evolution selection systems to generate ABE8e(TadA-8e V106W...alternative approach, engineering and directed evolution of the TadA domain has yielded base editors that...Tolar, J., Osborn, M. J., & Liu, D. R. (2023). Evolution of an adenine base editor into a small, efficient...

...2020) Directed evolution of adenine base editors with increased activity and therapeutic application. Nature...primary human cells. Now, scientists at Beam Therapeutics have found a way to improve editing in primary...Image from Gaudelli et al., 2020. Directed evolution identifies adenine base editors with improved ... and Head of Gene Editing Technology at Beam Therapeutics, used a previously developed bacterial selection...ABE8.x”: “8” stands for the eighth round of ABE evolution and the “x” is the placeholder for the number ...editing specificity, for example for potential therapeutic approaches, the authors recommend mRNA delivery...off-target deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved...

...proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These...et al, 2017). To add insult to injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming...Identifying PAM-free and -flexible Cas enzymes   The solution to the PAM problem seems simple, in theory: find...options, none of them offer a one size fits all solution to the issue. Nor are all Cas enzymes equal; just...generated by the Liu lab through phage-assisted evolution, specifically with the goal of generating Cas ...-less? Have we reached an essentially PAM-free solution? Almost! Assuming a PAM of NRN, 50% of randomly...History of CRISPR Cas – A Tale of Survival and Evolution  ...

...used as a guide to point you toward a creative solution where everyone wins. What are the underlying...Importantly, as the third example shows, the ultimate resolution can involve ideas not related to the actual data... beneficial to both.  Coming up with creative solutions where everyone wins can initially feel more difficult... get unstuck and move closer to an innovative resolution. Creative negotiation can also decrease or entirely...with negotiating (check out more about conflict resolution). How to develop negotiation skills You can easily...being remembered as a person who tries to find solutions that benefit everyone. Even if I “lose” a negotiation...understand my position and to find a creative win-win solution. What does professional negotiation look like?...

...cells (9). PA-FPs have helped to revolutionize the field of super resolution microscopy. PA-FPs, especially...reversible type, are very well suited for super resolution imaging, where the power of the technique relies...reconstruct an image. The best PA-FPs used for super-resolution imaging usually have the following characteristics-high...wavelengths. Indeed, within the world of super resolution microscopy, PA-FPs have helped create several... improved over the conventional FPs for super-resolution imaging, they still do not emit the large number...being engineered. By combining PA-FPS with superresolution microscopy, scientists now have at their fingertips...V., et al. "Photoactivatable mCherry for high-resolution two-color fluorescence microscopy." Nature methods...

...typically load 3 dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall within...The process to titrate AAV by ddPCR begins with diluting the virus. It is important to note that the dynamic...:6 million to 1:25 million.  After making the dilutions, they are transferred to a new plate containing...reduction in the number of positive droplets as the dilutions increase. The ddPCR software uses the ratio of...Copies/uL V = Volume of virus in reaction mix D = Dilution factor of virus Most droplet readers have a few...separate area from where you prepare your sample dilutions. Aliquot all of your reagents into single-use ... pipetting too quickly when making your virus dilutions. This will reduce aerosols that could potentially...

... protect you and your institution, and almost all universities and institutions require them for material...protective of both the providing institution and the receiving institution. Therefore, many technology transfer...benefits for both the providing and receiving institutions and their respective researchers. Check out ...knowing your rights – and the rights of your institution – are protected while we handle all the paperwork... to you, or we can send it directly to your institution’s MTA coordinator. Organizations that have opted...

...-based directed evolution approach with some notable improvements. Directed evolution techniques mimic...you'll find in this post: Yeast-based protein evolution Fluorescent tools for mammalian cells Nanobody... Faster catalysis after yeast-based protein evolution By Joanne Kamens Mateo Sanchez and Alice Ting ...time modulation of the TEV cleavage and improve utility of the system for use with low affinity variants... new system has been demonstrated to work for evolution of other target proteins and might be useful for...screening systems at Addgene! Schematic of the evolution platform to generate TEV protease variants. Catalysis...transcription of the mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS...

...the location and distribution of synapses in the mouse brain. Kim et al is utilizing a mammalian GRASP...random mutagenesis in the soil bacterium Pseudomonas putida and demonstrate that they can successfully create...transport. pMitoTimer from Yan’s lab, which is constitutively expressed, reports on the balance of biogenesis...constructed using Golden GATEway cloning have been utilized to create recombination template vectors, to perform...chemogenetic technologies After several years of distributing his Designer Receptors Exclusively Activated...cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly steps. Before beginning... (SSA) assay. This new Platinum Gate TALEN Kit utilizes a 4-module assembly system, which reduces the ...

...protein engineer currently working at Sanofi Pharmaceuticals in Cambridge, MA. When I joined Sanofi/Genzyme...including Activin A. A commercially developed therapeutic antibody that binds Activin A and interferes ...This is why we cannot stop searching for novel solutions to treat rare diseases like FOP until a cure is...fight against FOP in two ways. First, Addgene is distributing ACVR1 Plasmids from a variety of research labs...will be hosting the AJ’s Boston League team (Contributing many new heroes to the team in the process) ...protein engineer currently working at Sanofi Pharmaceuticals in Cambridge, MA. His work experience is in...in both vaccine subunit design and therapeutic biologic design (ADCs) along with pre-clinical analytical...

...Quality at Dimension Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of the... the original employees at Dimension Therapeutics, she helped build and grow the gene therapy division...research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several years ago through...Stegmeier is the Chief Scientific Officer at KSQ Therapeutics and is an Addgene depositor. KSQ uses a CRISPR-Cas9...Barcoding Library, a library that “enables the high-resolution tracking of more than 1 million cancer cells ...himself to finding “immunological and virological solutions to crises like those and ended up in the field...

...caspases, protease enzymes that cleave proteins. Executioner caspases are activated last in the cascade and...scientists have used genetically encoded fluorescent executioner caspase reporters. Several apoptosis reporters...Forster resonance energy transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET... an improved apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11... of the linkers is a consensus caspase-3 (an executioner caspase) cleavage site (DEVD). Upon cleavage ...of sfCherry approximately 20 fold by directed evolution. They then optimized the length of both linkers...fold increase in fluorescence after cleavage by executioner caspases. Failure for cells to die or cells dying...