...enzymes   The solution to the PAM problem seems simple, in theory: find a Cas enzyme or evolve one that...Cas proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs...engineering of the PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed the SpRY...display higher off-target effects than SpCas9, a pleasant surprise since the PAM flexibility could in theory...

... around the world to detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield...antibody expression. If starting from a protein sample (for instance, affinity purified antibodies from...recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. eLife 8: . https://doi.org...NH, Rahman MZ, He L, Xin L, Shan B, Li M (2016) Complete De Novo Assembly of Monoclonal Antibody Sequences...

...different activation/inactivation kinetics. For example, the light-gated cation channel, Channelrhodopsin...Therefore, light must be delivered in pulses. An example of a standard stimulation parameter is 20Hz (laser... control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells... Central PMCID: PMC3769938. 5. Iwai Y, et al. A simple head-mountable LED device for chronic stimulation...

...alternative PAM sequences are longer and often more complex than NGG, resulting in lower overall genome frequency...According to this logic, ω-dCas9 variants recognizing multiple PAMs should have a fitness advantage over proteins... and CAA PAMs, they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (...modifications that can be made to Cas9. It is a pleasant surprise that selecting for one beneficial trait...

...site destination vectors for inserting a tag at multiple target sites. SapTrap vectors can be easily modified...Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein... assay additional protein characteristics. For example, Masato Kanemaki's lab has developed a CRISPR/Cas-based...PMC4974478. Natsume, Toyoaki, et al. “Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging...

...where the PhD application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where...of schools by meeting with professors - a/k/a Principle Investigators (PIs) - whose labs does research...right fit. Reaching out to PIs also means doing plenty of mini-interviews; these professors will not only...campus even if you can’t visit. By talking with people at the institution, you can get a better sense ...

...fluorescence when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence ...intensity decreased whenever the magnet approached the sample (by less than 1%) and recovered after the magnet...interference, but the result persisted. (Even better, multiple other labs have reproduced their EGFP findings...expressing MagLOV while a magnet is waved under the sample. The imaged area measures approximately 82 mm across...

...signals are integral to many facets of biology. For example, many heart issues stem from the heart beating ...which are electrical charges propagated through multiple cells to do things like translate perceived signals...naturally occurring voltage indicator. To give an example, a genetically encoded phosphatase (voltage sensing...then localize and emit signal from. This specific example is attractive due to the large number of sheer ...

...selecting plasmid-containing bacteria. So… why do some people still use amp? Carb is expensive (relative to amp...host/plasmid DNA during selection if it’s not completely bound up. While this likely occurs at a low rate...attention to antibiotic selection. If you feel like multiple of the above antibiotics would work for you, they...Antibiotic Resistance Stability Time Saving?  Works for multiple organisms? Compatible with most bacteria? No satellite...

...way to increase CRISPR editing efficiency across multiple CRISPR enzymes. Plasmids for CRISPR coselection...was also independent of another TTAGGG binding complex, shelterin. Swapping the DNA binding domain of ...segment on regulating telomere length with TZAP Multiplexible plant genome engineering toolkit The Voytas ... well as Cys4, tRNA, and ribozyme systems for multiplex gRNA expression. Expression of toolkit components...that are sure to be enabled by this toolkit. For examples of the toolkit in action, see Cermak T, et al....

...the American Chemical Society (ACS) are just 2 examples of organizations that hold large, annual conferences...that did the same thing? Make an effort to meet people. Attend smaller talks within the bigger conference...professionals, with the added benefit that most people at the fair will probably be willing to accept ...country; sometimes several per month, giving you a plethora of opportunities to explore! Again, attending ...

...Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall... Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science...https://doi.org/10.1126/science.aaq0180  Fine EJ, Appleton CM, White DE, Brown MT, Deshmukh H, Kemp ML, Bao..., Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156:935–949...

...only occur through editing by a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1:...DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create...DNA editing efficiency of APOBEC-Cas9 editisome complexes. The eGFP reporters rely on the correction of ... single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: . https://doi.org/10.1038...

...become resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma species...Figure 1): Over-prescription of antibiotics (for example, giving antibiotics for an infection that isn’t...treatment regiment might wipe out bacteria that are completely susceptible to a drug while a full course of ...likely to develop over time. It isn’t feasible to completely eliminate the development of resistance; it’s...

...We think it’s so important that we have written plenty of articles on building successful mentor relationships...experience In research there is a saying that if the sample prep is poor, then the data will be poor. The same...you remember where the equipment, reagents, and samples are. Do: Ask your bench mentor if they have time...your mentor know what yours is! For instance, some people like to ask questions throughout training, while...

...tool is in such a small package. But don’t let the simple presentation deceive you; pooled libraries can ...visualization) that confirm diversity of the library sample, and any other information or scripts provided ...suffice…and the answer is no. Due to the size and complexity of pooled libraries, NGS is the best way to validate... plasmids didn’t amplify after your screen is completed!  Producing Your Lentiviral Library Once you've...

...discovery can be hard since it’s frequently why many people get into science in the first place. The feeling...but for many graduate students and postdocs, the people you see in your bay for 8-12 hours a day become...worked at the bench for over 10 years, but when people ask me about my job, I have no qualms about saying...course, but you’ll inevitably have downtime and plenty of it. That’s not the case at an office job. I’...

... and the complexity of effectively deploying them in the lab somewhat mirrors the complexity of understanding.... Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult both to...broadly useful vectors, a process that isn’t as simple as it seems. Take, for instance, the GCaMP-encoding...

...camera or tracking software. Locomotion is a highly complex process on the level of individual neurons and ...motivational and other aspects may be involved. For example, an animal moving around to explore the environment...the elevated plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and ...behavioral neuroscience to reveal insights into the complex relationship between genes, neurons, and behavior...

...Amplified nucleotides are combined with Cas 13a in complex with crRNA targeting the sequence of interest and...sequences from a population (either enriching or depleting specific RNAs out of a pool of RNAs) or studying...cleavage system. LwaCas13a is also amenable to multiplexing; expressing 5 gRNAs simultaneously produces ... Featured Topic Page Learn about Anti-CRISPRs Multiplex Genome Editing with Cpf1 Resources on Addgene.org...

... into one of the loop regions of any G-protein-coupled receptor (GPCR) recognizing a specific ligand (...into an intracellular loop of various G-protein-coupled receptors. Image adapted from Ino et al. 2022, ...with different ITAM/SH2 reporter pairs allow multiplexed measurement of different RTKs. Although this ...to the range of concentrations expected in your samples. Higher affinity isn’t always better, especially...

...or entire organism. Because each cell can have multiple copies of the transgene that will recombine independently... all cell types can be labeled, and that while multiple copies of the transgene can integrate into cells...dermis and identify that Langerhans cells are replenished through the proliferation of specific, and not... neurons over millimeter-long distances across multiple fields of view, whereas previous Brainbow technologies...

...are essential cell surface receptors involved in complex signaling pathways and the linkage of intra- and...proved challenging to study. Cells often express multiple integrins that bind the same ligand, making it...development. Integrin structure and conformational complexity There are 24 known mammalian integrins, all heterodimers...demonstrated that anti-integrin αVβ5.9 “essentially completely blocks all adhesion,” inhibiting the function...

...plasmids with the piggyBac™ transposase itself, please read on to learn more about this exciting technology...also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations...challenges still remain, such as making gene edits completely seamless (i.e., so no other genetic alterations...cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.,” Proc. Natl...

...New to the lab? Or looking for ways to supplement your teaching? You’re in luck! Addgene’s Intro to the...vectors, but what we lacked were videos guiding completely new members of the lab or those taking introductory...techniques. It’s also great for teachers looking to supplement their curriculum. From the details about personal...

...tens of thousands of biomolecules each containing multiple binding crevices that can nonspecifically soak...measureable FRET will be swamped out by a sea of uncoupled donors. The best method for targeting a fluorescent...NTA binds to the His tag on your protein and the coupled Cy fluorophore acts as the FRET acceptor. Binding... calibrated FRET distance measurements (for an example see Mahalingam et al., 2014).  Many thanks to...

...lab? Addgene is after all a place that encourages people to take some time out every once in a while – just... stress in adolescents. It also put those young people in a better mood. Laughter itself can sometimes...and, of course, it’s a way to connect with other people. (We don't need evidence for that one, do we?) ...

...export controls and regulations are complex and may change suddenly. Please email [email protected] for the ...If you have any questions on ordering plasmids, please feel free to email [email protected]. We’ll be happy...

...Watson & Crick - Science History Rap Battle A great example of science story telling following the unfortunate...importance in understanding biological history. 4. Please Don't Kill My Hive Beautifully filmed video about...science educator looking for creative ways to implement the next generation science standards and communicate...

...sustainability effort we’ve implemented at Addgene. The kitchen and office areas provide multiple streams for composting... new programs, we're always striving to set an example of environmental stewardship.  Additional resources...

...pharmaceutical company based in Switzerland. After completing her PhD in Chemistry and Chemical Engineering...working in industry and how delegating can empower people Chapter 3: How Crystal advanced at Novartis, and... and the importance of realizing that different people have different management needs. How Crystal developed...

...single base changes in the DNA sequence without completely cutting the DNA. RNA Editors: CRISPR nucleases...CRISPR nucleases that can’t cut DNA (dCas9, for example) that are used to increase or decrease expression...library of gRNAs with a CRISPR nuclease target multiple genes in a cell population. The resulting mutant...

... they have developed this further to provide multiplexed detection, higher sensitivity, and more. Read...lab compared the efficiency and specificity of multiple wild-type and engineered variants of Cas nucleases...included in the next What’s New in CRISPR blog post, please let us know! Additional resources on the Addgene...

...gene/target, name, application, pass/fail rating, sample species, antibody species, or whether the results... by Addgene's curation team for accuracy and completeness. The information is then formatted and published...application. The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate...

...looking to delve deeper or stay tuned, there are plenty of online resources available. I recommend a few...Science Translational Medicine have made their complete collection of relevant papers freely available...you have information or another resource to add, please tell us about it in the comments. ...

...template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease PCV. This increases...Neisseria meningitidis, Nme2Cas9, that recognizes a simple dinucleotide PAM (N4CC), lending to high target...included in the next What’s New in CRISPR blog post, please let us know. We also welcome blog posts from guest...

... Aavery in this Zoom video background. Want the purple bacteria video background with mascots Blugene ...and more!) on Addgene’s Help Center. Download the purple bacteria background.       Are none of those ...Addgene’s Science Careers blog topic. Download the purple lab-themed background.     We hope these science...

...results Above (top right corner of this post) is an example of a seemingly clean DNA sequence (no Ns in sight...less and you might suspect contamination in your sample or consider asking your sequencing facility to ...Use a silica spin column for purification of the samples you send for DNA sequencing If your sequencing ...

... of the coast of Washington state. Using these samples, he was able to isolate two proteins from the jellyfish...potential as a research tool. Soon, the number of people studying it grew. In particular, studies in the...organisms to find the culprits. He even obtained samples of certain ones from local pet shop aquariums in...

...Stefan Stricker’s lab, is a single step gRNA multiplexing system that generates vectors containing high...efficiency can be controlled by manipulating complementarity between the gRNA and target. Read the paper...included in the next What’s New in CRISPR blog post, please let us know! Additional resources on the Addgene...

...scientific community? Access to plasmids which gives people more time to focus on their research.  How will...plan for moving forward. Sooner or later enough people will be vaccinated and we’ll have some herd immunity...raises questions like “How do you start to bring people back to on-site work?” and “How do you start to...

... need a high efficiency of transformation (for example, if you are working with a library or an especially...making competent cells are available online. A few examples are provided here for reference: OpenWetWare ...transformation. This protocol has worked well for us for multiple E. coli strains, including DH5alpha, XL1 Blue,...

...cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available at Addgene, also...tested GreenGate in Arabidopsis, it should in principle work in any plant species. Tricks of the trade...essential for scientific progress. Also, the more people use such a system, the more building blocks will...

...interviewing fellow Addgenies. I spoke with amazing people that were a part of the different departments of...felt like I could actually make connections with people. These were some of the things that really made...by Addgene. I have met some of the most genuine people who have shared amazing advice with me. Due to ...

...team costumes, and spooky science stories. Yes, please! At Addgene, we love finding ways to incorporate...bird feces, which get eaten by another snail, completing its lifecycle. Halloween genes The Halloween ...and drains all the blood out. Once the blood is depleted, the tongue atrophies and falls off. The louse...

...Science Communication group started. It's as simple as getting people together at a bar. Chapter 10: Maintaining...well as a platform for scientists to be viewed as people (#ActualLivingScientists). We also discuss the ...

...should receive the clinical sample to test their phages. Because the sample is limited, only the labs that...targeting organisms closely related to the clinical sample. For treatment, it is best to identify a cocktail...

...affected by devastating diseases that affect far fewer people and therefore fail to garner much international...the Institute of Biosciences and Bioresources in Naples, Italy. His group works on spinal muscular atrophy...data from primary peritoneal mesothelioma tumor samples that they have used to identify candidate genes...

...website as a “Publication Citing this Plasmid” - this simple act directly associates you with a network of researchers...functionality can be found in the following video: Please remember that the accuracy and reliability of our... plasmid is missing from our collections pages, please shoot us an e-mail at [email protected]. We’d love...

...YFP) to the new and improved fruit colors (e.g., apple, papaya, and tomato) and the photoactivatable fluors...quickly. Researchers should keep in mind that a very simple sequential or double restriction enzyme digest ...on the corresponding plasmid pages (e.g. see Supplemental Documents here). If you’re not sure where your...