...assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology ...cloning method, but it is limited by its sequence constraints. The long overhangs must not contain the dNTP...ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination...the linearized vector by T4 digestion; then the components are combined and annealed. The resulting recombination...up a new world of multicomponent assembly, as fragments with overlapping sequence homology can be assembled...of multicomponent assembly with SLIC. Multiple fragments are PCR amplified to add 5′ and 3′ homology regions...vector. 5′ overhangs are generated on all the fragments and the vector, specifying the order of assembly...

...few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenence within an E. ...different cell types and each one requires different elements for vector propogation. This post, along with ...and explain how they differ from the bacterial elements previously described. Why do scientists use yeast...complex internal cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally...in the lab typically contain all the necessary components to allow shuttling between E. coli and yeast ...the bacterial cell. Similary, the specific ORI elements included within a yeast vector determine how the...replicated and maintained within the yeast cell. These elements control not only the number of plasmids found ...

...functions. Experiments come next, testing the correctness of the hypothesis. The results of experiments can ...already encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew...Politics and science are intrinsically linked: governments exist in reality and science is how we learn ... to protect their rights (2). Less formally, governments are the organizations that make and enforce the...information about reality informing people and their governments on what outcomes are actually possible for a ...informed fashion. For science to be useful to governments, however, inconvenient data must not be ignored...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...

...the FP(11) and FP(1-10) fragments. When Protein A and B interact, the FP fragments can assemble the full...al. 2017 under CC-BY license. Many split-FP fragments are named according to the number of strands in...10 and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be ...be compatible with multiple FP(11) fragments, even from a different base FP (for example, EBFP2(1-10) can...Venus Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis...pBiFC-VC155 Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis...Chang-Deng Hu mVenus pET-BiFC contains both fragments of split mVenus (aa 155), includes I152L in N-...

... the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases through...group recently reviewed and reported that PMM2-CDG patients suffer from immunological defects. These are characterized...almost every aspect of immunology, participating in events such as cell development, recognition and migration...migration, the immune compromise observed in CDG patients is not surprising. However, the molecular and cellular...cellular mechanisms behind the immune defects in CDG patients are still poorly studied and understood.  In line... to lower the burden of immune symptoms on CDG patients and to increase their quality of life by reducing... the lab will be greatly facilitated by CRISPR reagents and other specific gene sequences available from...

...PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there are ...most gene targeting experiments. Between SpRY and several of the other Cas variants described, nearly all...need for gene editing in research, the old PAM constraints just won’t cut it anymore. Say hello to PAM-free...PAM must be selected. When it comes to CRISPR experiments, therefore, you often must make a choice about...region will do the job. What matters for these experiments is cutting efficiency so that the greatest effect...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative... generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to...

...for high-quality antibodies and reagents that work for our experiments!)     Figure 1: The IBEX...their successes (recommended reagents) and failures (not recommended reagents) along with information critical...tissue preservation method, and detergents that they used in their experiments. Researchers can then visit...interested in having high-quality antibodies and reagents that you know work…preferably before you start...dataset currently includes 1049 community-validated reagents from 20 contributors, including 61 fluorescent...works and in what doesn’t, and feedback are vital components of building both the community and the database...Community’s vision is an open, global repository for reagents, protocols, panels, publications, software, and...

...CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method was published...CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original CAPTURE...of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA...genomic location of interest  When these three components are co-expressed, the gRNA targets the dCas9 ...then developed by detecting these chimeric DNA fragments by PCR or next-generation sequencing. Figure...diagram). Cross-links are reversed so that DNA fragments can be detected by PCR or deep sequencing. ...number of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells...

...organizing events. I contacted local professional organizations and nonprofits. I volunteered at events like...resources daily to promote CINSO events and recruit speakers and participants for our professional development...Science Outreach, or CINSO. We organize fun science events for adults and professional development opportunities...best I began viewing my activities as types of experiments. I searched for new opportunities with an open...wasn’t something I liked – well, I was used to experiments not working, so just like in lab, I sucked it... – call it “troubleshooting.” Trying out new experiments and new ideas is nothing new to a scientist. ... opportunities provided by your programs and departments Graduate school was an excellent time to try ...

... a library of Arch mutants screening for improved brightness. The brightest mutants were then further ...from the past year? Let us know about it in the comments or shoot us an e-mail at [email protected] and ...Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA cleavage upon co-expression... of specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES... of recording permanent "memories" of molecular events. These memories would be recorded in the cell's...the relationships between pH changes and cellular events. To overcome the issues with fluorescent pH sensors... tools for spatial and temporal control of cell events Three years ago, Brian Kuhlman’s lab designed photoswitchable...

...data with you. “Affinity reagents are among the most commonly used reagents in neuroscience research ...to introduce affinity reagents in protein format within the next year. NIH grants and Addgene’s mission...focus of the NABOR platform will be neuroscience reagents and data, the platform can be expanded for any...secrete antibodies in culture and provide renewable reagents with reliable and reproducible production. However...recombinant antibodies as well as other affinity reagents such as single-chain variable fragment (scFvs)...model for the creation and acquisition of affinity reagents and will enable researchers everywhere.” The ...door for a new generation of recombinant affinity reagents. Addgene is looking forward to working with the...

... - the tendency for students to be more interested in studying animals than plants, and biology teachers...many benefits. Engaging students in active learning through hands-on experiments allows them to develop...conduct the experiments outlined in ABRC’s kits vary by module. As a general guideline, experiments that include...concepts than can be gained from lectures alone. Using plants as an experimental system comes with fewer ethical...include growing plants require pots, trays, soil, fertilizer, seeds and a growth space with fluorescent...fluorescent lights. Other experiments require only seeds, petri dishes, and filter paper. Another benefit of using...questions about growing conditions, to guiding students through specific protocols, ABRC staff are available...

...because when RSpace documents are deposited into the repositories, links in the documents to data in the external...reproducibility RSpace was designed in response to requirements that emerged from intensive engagement with ...relevant to a particular experiment, series of experiments, or project. ‘Small’ data produced by the research...lab servers specifically designed to hold large amounts of data. Although it may be useful to selectively...reproducibility. For example, it’s possible to import Word documents into RSpace, maintaining the original formatting...edited, and it’s possible to link between various documents. A second example is the integration between RSpace... a two-way integration. It’s possible to send documents from RSpace to a selected Slack channel for discussion...

...LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments open up new possibilities...vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp smaller than previous...PE6d are optimized for dual-AAV systems. These variants use different RT enzymes and performed large edits...such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...interchangeable set of fusogenic and targeting components to reprogram delivery vehicles like lentiviral...DIRECTED particles where the fusion and targeting components are interchangeable. Image from Strebinger et...demonstrated LOV-Turbo in neurons, in pulse-chase experiments, and more. Find LOV-Turbo plasmids here! Next...

...the PAM Requirements of Different Cas9 variants Learn about CRISPR Pooled Library Experiments Additional...even image your favorite gene. With so many Cas9 reagents available, it can be difficult to decipher which...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...modifications Site-specific deletions and knockouts Some experiments may require the introduction of a mutation at...an expanding number of Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement...Cas9 proteins (such as SpRY) with altered PAM requirements, giving it more flexibility. A consideration...target genes using dCas9-based repressors Early experiments using dCas9 in bacteria demonstrated that targeting...

...the game rules and then you let your students play. As your students play, you can observe, answer questions... make our classes more practical and hands-on. Students in these courses get plenty of theory in their... genetic manipulation of human stem cells when students are not even allowed to manipulate prokaryotic...cells? Especially, if you only have 4 hours and 35 students! After a course coordination meeting with some... I didn’t want to face another group of sleepy students at 8 a.m. many of them focused more on social ...therefore started planning a game that would force my students to use concepts learned from previous lectures...win-win strategy. After each team presented, all students had to pick the best strategy and propose a final...

...labor with up and coming immunotherapy treatments. As treatments enter the clinic, physicians and researchers...keep ahead of the disease in patients and developing collaborative treatments will go a long way in combating...macrophages (M1 and M2) that help create welcoming environments for tumor cells spreading to other organs during...to inhibit further cancer growth and subjecting patients to the risk of microbial infection. Chronic inflammation...one of the most commonly used immunotherapeutic agents. Currently the FDA has 12 approved monoclonal antibodies...cells and T cells activated ex vivo are also contestants for cell-based immunotherapy. Using activated... CRISPR-based clinical trial in 18 lung cancer patients. This study used CRISPR gene editing to knockout...

...microenvironment, a tumor’s backstage team. Microenvironments are cellular regions with specialized structures...structures. Tumors are surrounded by microenvironments and what encircles a disease is highly prone to affect...precisely that for breast cancer. What are tumor microenvironments made of?  A tumor’s genesis is divided into...development? Being pathological active regions, microenvironments shape a tumor’s evasiveness, evolution, and...others repress it. Unfortunately, as a whole, microenvironments promote tumor durability instead of repressing... To understand how cancer cells influence microenvironments and vice versa, let’s take a look at cancer...improving cancer cells proliferation.  How do microenvironments affect tumor spread and drug resistance? Although...

...from their contents. You should be tracking both, you should easily be able to move contents from one container...can be useful in academic labs where postdocs, students, and lab mates are coming and going frequently...Addgene website, it sets off a chain reaction of events throughout all of Addgene - within the office, ...applied to containers and not the mushy, gooey contents of said containers, think carefully about how ...encoded numbers. Loosely couple containers and contents Right from the get-go be as clear as you can be...are being applied to the containers, not to the contents. Tightly coupling barcodes and containers might...such a bad idea, tightly coupling them to their contents might leave you sad and confused when a tricky...

...1-3 kb (approximately gene-sized) fragments so that these fragments can be cloned into E. coli and subsequent... Express random phage DNA fragments in E. coli Now that random DNA fragments have been cloned into E. ...return no candidates. Our strategy: Express random fragments of the phage genome and probe with live cells ...DNA frequently enough to result in a smear of fragments that get smaller in size as a function of digestion... method that results in the bulk of resultant fragments falling between 1 and 3 kb results in optimal ... causing issues associated with cloning large fragments. After fragmentation, repair sheared ends with...into chemically competent E. coli and analyze transformants for appropriately sized inserts. We consider...