...standpoint, a plasmid with all four genes and necessary control elements for each would be relatively large. Cloning...separate plasmids allows researchers to partially control expression levels of each gene by altering the ...genes (either viral genes or minigenome) are under control of a T7 RNA polymerase promoter sequence. Thus,...

...comment, annotate, and edit protocols. After my introduction to protocols.io, I decided to research what ...orders, tracking students and research projects, electronic lab notebooks and protocol management. Though...More Reading: How to manage scientific data: Electronic lab notebooks compared with Google Docs and wikis...

...provided a major toehold into understanding how genes control biology (a fact celebrated by the Nobel prize committee...transgenics Creating transgenic organisms by introducing foreign DNA into the genome is useful for a variety...into adults and explain how signaling pathways control this process. Additional zebrafish applications...

...advantages. An introduction to Gateway technology The Gateway cloning method, developed by Invitrogen, is an ...an in vitro version of the integration and excision recombination reactions that take place when lambda...

...laboratories and associated publications, but our quality control (QC) standards have evolved over the years to keep... ability to re-use or borrow existing code and control how the various pieces were connected, as this ... for Bioinformatics AnalysesAAV Vector Quality Control: Going the Extra Mile with NGS...

...a large number of requests for vectors in the “controls” category, which includes things like viral vectors...vectors in a variety of serotypes, including the retrograde AAVrg and the systemic capsid PHP.eB.   ...Patrick, M., Guerin, K., & Fan, M. (2023). Quality control for Adeno-associated viral vector production. Neuromethods...

... for saturation mutagenesis, and they provide a strong proof of concept for further development. ...excision repair pathway. The team behind AYBEv3 introduced the first deaminase-free base editor when they... called gTBE and gCBE, with Addgene. gTBE can introduce T → C and T → G edits, though with limited selectivity...

...degraders. But CRBN has been difficult to study in vitro, requiring either expressing it with additional ...codon-optimized for C. elegans and included artificial introns to improve expression. When using these constructs... an edited strain carrying Cas9 to allow the introduction of additional sgRNAs. Image reused from Neo ...

...of symptoms in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy...therapy. In 2014, RSRT brought together a strong collaboration between two gene therapy labs (Brian Kaspar...neurological disease (Sinnamon et al., 2020). They introduced a mouse MECP2 guide RNA and a hyperactive version...

...protein instead localizes to the cytoplasm? Or more troubling, what if a significant fraction of the fusion ...these compartments using CRISPR systems should strongly consider using moxFPs over standard FPs for the...Structural Biology. His interests include the quality control of secretory proteins in the Endoplasmic Reticulum...

...epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure...determination By Mike Lacy Conventional cryogenic electron microscopy (cryo-EM) protocols typically require...glia-specific intermediate filament expressed in astrocytes throughout the brain, making it a popular target...

...pGP-AAV-syn-jGCaMP8f-WPRE (AAV Retrograde) pGP-AAV-CAG-FLEX-jGCaMP8m-WPRE (AAV Retrograde) pGP-AAV-syn-FLEX-jGCaMP8m-WPRE...have Kd around 70 mM and 100 mM, respectively. Introducing mutations and using potassium-binding protein...

...handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... Addgene as a source of sequenced and quality controlled plasmids and viral vectors. When working with... easier to learn new experimental techniques. Electronic lab notebooks also play a role here by making...

...transcriptional regulation, as long as they can control recombinase activity. For example, post-transcriptional...methods section of our paper (1). How might one troubleshoot GSRMs that don't work as expected? The successful...the parts used to build them. We recommend using strong, directional terminators and promoters with buffer...

...the number of integration copies can be tightly controlled by titrating the transposase to transposon ratios... levels of your gene of interest, as needed. Controlling the expression levels of your gene may be particularly...splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.,” Nat. Protoc., vol. 4, no. ...

...and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs through Golden Gate cloning. Artificial...Identifying actively silenced plants Once you’ve introduced your RNAi into your organism, how do you identify...insertion of any target sequence, as well as essential control plasmids. Many thanks to our guest blogger Robert...

...interaction is not covalent, later work showed that introducing a Leu28Cys mutation in eDHFR does lead to covalent...2). If you’re working with purified proteins in vitro, you can even use the same tag for multiple proteins... actin filament assembly-regulating proteins in vitro reveals complex events. a) Micrographs of a fluorescently-labeled...

...CRISPR/Cas9 allows us to delete sequences and introduce mutations and epitopes with unprecedented ease...in 1-2 kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette... ML, Jorgensen EM (2018) SapTrap vectors for introducing point mutations with unc-119+ selection. https...

...happy cloning! This article was written by Susanna Stroik and Meghan Rego, with thanks to Rob Hurt for his...Plants Mediated by a Cas9 Gene Containing Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier...Matthew S., et al. Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells. Cold Spring Harbor ...

...substance (fluorophore) that has absorbed light or electromagnetic radiation at another wavelength (Heinrichs,...Heinrichs, 2009). When a fluorophore absorbs light, electrons are elevated from a ground state to an excited state...After a brief elevation in this excited state, the electron returns to its ground state by emitting a photon...