...overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single ...increased number of fragments, or the ligation of very small/very large fragments, these problems can ...homology at the ends of DNA fragments to specify assembly order, so fragments with 5’ or 3’ sequence homology...they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites... These overhangs specify the desired order of fragments, and the loss of enzyme recognition sites after...BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. As with all Golden Gate-based methods...outside their recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled...

...site-specific recombination events. The system consists of two components derived from the P1 bacteriophage...present; intramolecular events happen with greater frequency than intermolecular events, but any two sites ...series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags...recognition site. The P1 bacteriophage uses these components as part of its natural viral life-cycle, and ...and researchers have adapted the components for use in genome manipulation. Cre recombinase, originally...allowing for broad applications in a variety of experiments. LoxP (locus of X(cross)-over in P1) sites are...modification can be restricted based on experimental constraints. This has been used for a wide range of purposes...

...money on optimizing experiments, to false conclusions.  Figure 1. Potential points where DNA errors...shifts, missing elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences...an important part of maintaining the quality of reagents, especially shared ones.  The importance of QC... propagation of plasmids in bacteria — has many points at which small errors can be introduced. Eventually...introduced, indicated by exclamation marks. Error points include places where DNA is being created, such...tags, promoters, and other critical functional components. Fun fact! Some older plasmids in our collection...each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated...

...link together multiple fragments, allowing ordered assembly of multiple components into a destination vector...suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...(here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly into a Cas9...and thus the highest frequency of genome editing events. Yamamoto Lab Multiplex CRISPR/Cas9 Assembly Kit... with the Gersbach lab plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase...clone your own entry vectors containing other Cas9 variants to use with the MuLE system. Multiplexing from...are also available from the Yang lab for use in plants. The mammalian multiplex systems use the Csy4 RNA...

...can vary "Lab assistants" usually wash glassware, prep media, etc. "Research assistants" are more involved... researchers, as well as graduate students (your teaching assistants), about their experiences. Research...investigator (PI) Runs the lab Writes a lot of grants Varying degrees of engagement with what happens...degree "Rotation student" - some PhD programs have students rotate through a few labs, ~10 weeks per lab, ...way to answer those questions. I’ve met graduate students who enjoy the thrill of acquiring a piece of knowledge...with your labmates and mentors Many of the grad students and postdocs I’ve met have done things that I ...about careers in science are geared towards grad students and postdocs—people who are a little ahead along...

...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...