...cloning: A SLIC makeover In 2007, LIC received an important update, courtesy of Addgene depositor Stephen ...the linearized vector by T4 digestion; then the components are combined and annealed. The resulting recombination...BioRender.com.   SLIC also opened up a new world of multicomponent assembly, as fragments with overlapping sequence...efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple fragments are ...issue is sequence similarity. If fragments in a multicomponent assembly have 5′ or 3′ sequence homology to...

...attended a scientific symposium called Horizons in Molecular Biology. This annual symposium is organized by ...designated work areas and come with health risks. Importantly, with this method it is not possible to follow...Chemistry, Göttingen in Germany. I attended this symposium seeking inspiration to resolve the technical hurtle...FUNCAT-PLA provides both quantitative and spatio-temporal information about the turnover of specific endogenous...

...any other important features that differentiate this plasmid from its predecessor. Important features ...restriction sites used during cloning, and most importantly, the predicted full plasmid sequence. Other information...sequence verify the plasmids. This is the most important step in ensuring quality, as it will catch any...sometimes have to design new primers to sequence the important features. You can use primer design software such...

...co-coregulator) partners. The coregulator ‘core’ complex is composed of a tightly-bound invariant group of proteins...comprehensive datasets that collectively address many important and fundamental questions in nuclear receptor ...states, their circadian regulation, as well as in important physiological states such as adipogenesis and ...for a reasonable fee. We at NURSA recognize the importance of this service to the NR research community ...coregulators. We look forward to growing this important relationship with Addgene for the benefit of the...

...the following four components into 293T cells. Figure 1: The key components of Nanoblades. Image...CRISPR applications. Let’s take a look at the key components of Nanoblades, how their editing efficiency and...for HDR editing and mouse-model generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery...transgenic mice by microinjecting zygotes with CRISPR components. However, this sometimes requires intracellular...

...length can and should be varied as it can have important implications for the fusion protein effectivity...included for visualization purposes, but the key component of the editing assemblies are sensor-actuators...upon these original tools that allows for both spatiotemporal protein degradation and quantification of this...expressed under a desired promoter with a specific spatiotemporal expression pattern and an auxin inducible degron...Resources page.  Plasmids for delivering CRISPRi components by a single AAV vector are now available from...

...specific tissues and cell types. If desired, spatiotemporal control of transgene expression or activity...effective in both mouse strains. It is therefore important to review the literature for use of a capsid in...Monkeys and rodents. Image and caption adapted from Campos et al., 2023. AAV.CAP-Mac is a systemic capsid...730825. https://doi.org/10.3389/fimmu.2021.730825 Campos LJ, Arokiaraj CM, Chuapoco MR, Chen X, Goeden N...Wang, T., Wolfe, D. A., Arokiaraj, C. M., Ngo, V., Campos, L. J., Griffiths, J. A., Ichiki, T., Mazmanian...Ravindra Kumar, S., Arokiaraj, C. M., Plattner, V. M., Campos, L. J., Mich, J. K., Monet, D., Ngo, V., Ding, ...Scherrer, J., Winchester, J., Blackburn, R. J., Campos, L. J., Man, K. N. M., Sun, J., Chen, X., Lefevre...

...how to prioritize but I  just added the “most important” tasks my schedule. This was bad for 2 reasons...expectations, answer any questions, and (most importantly) decide on a timeline. My experience is that ...your team’s success depend on it.  Equally as important is to be able to actively listen - this can be...for the best. Positive feedback is equally as important so don’t forget to recognize someone’s hard work...managers and I've learned from both. The former will empower you, keep you motivated and help you grow in your...

...generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters et al., 2019 with permission... Top: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes...ability to synthesize diaminopimelic acid (DAP), a component of the cell wall. Once in the recipient cell, ...Bottom: An ICE element containing the CRISPRi components is transferred to recipient bacteria by bi-parental...experiments where maintaining library diversity is important, while lower transfer rates are only suited for...

...post was updated on Aug 20, 2020. What genes are important in your phenotype of interest? Many scientists...not entirely known. Identifying which genes are important for a phenotype can lead to a wealth of additional... and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease (Cas9) and a single...library is a complex mixture. Pooled libraries are composed of many different gRNA-containing plasmids, each...strong evidence that a particular gene is actually important for a given phenotype. Each hit from the screen...

...experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting... by commercial vendors such as IDT. These two components are then incubated together to form the RNP. ... account with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be...scientists with an additional means to deliver CRISPR components to their experimental systems of interest. Using...it is likely that the use of RNPs will play an important role in its continued advancement. Andrew Hempstead...

... a Type I-F CRISPR-Cas system. The four major components required for RNA-guided DNA integration are (...sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon system... proteins (TnsB and C). While requiring fewer components for RNA-guided DNA integration, Type V-K systems... promoters were used to express the necessary components in E. coli (Klompe et al., 2019). Recently, the...simple to use, users should keep aware of a few important details. For transposons to be specifically recognized...

...of the vector components are HIV-based? A7: No single plasmid contains all the components necessary to ...The components are divided as follows: Transfer vectors contain minimal cis-acting HIV components: LTRs...LTRs, PPT, RRE, and psi packaging signal. Viral components typically total <1.5kb, which is rarely more ...

...of production have also made aptamers an important component of high throughput assays with microarrays...diagnosis. As biosensors, aptamers hold a few important advantages over their amino acid-based counterparts...laser cleavage and an RNA aptamer that bound the compound malachite green (MG) to create a non-protein based... 4-hydroxybenzylidene imidazolinone (HBI)-like compound allowing it to fluoresce, the Jaffrey lab asked...

...published data. The importance of data sharing The reason this topic is important to my lab is three-fold...their own data? A distinct (perhaps similarly impoverished lab) might apply different or novel tools and...work this may entail.  So if access to data is important, what data should be included? We should recognize...deliver on well-meaning but impractical requirements imposed by publishers and funders. Clearly, it is in our...

...likely not a coincidence that the AAV genome is composed of single stranded DNA, which is particularly ...with G418-selected clones. However, there are important disadvantages to the AAV-based approach that must...systems. Ohmori et al 2017 use a dual vector system composed of SaCas9 and a gRNA in one AAV and a repair template...researchers have devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied...2017 recently showed that by delivering CRISPR components as RNPs and delivering two halves of a repair...

...explaining my approach to conflict resolution, it’s important to recognize that there are different types of...or goal. Resolving these types of conflicts is important for the individuals involved, and also for the...That can mean a willingness to make yourself temporarily uncomfortable for the sake of a common good. ...with whom you strongly disagree, is a critical component of conflict resolution. Having empathy does not...transferring this skill to another job, it will be important to retain the stories and examples that have guided...

...this article, we’ll go through some of the most important considerations to make when planning and performing...same sample. For this strategy to work, it is important to ensure that you are using secondaries with ...primary, so we’ll start there. Some of the most important considerations are to make sure your secondary...publications or as a review on a company website. It is important to review the data and protocols available for...regardless of how “risky” they may be, it is always important to verify their performance in your hands and ...

...proteomics approach to monitor the composition of spatiotemporally defined protein complexes." Nature ...calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol reagent can be ... of exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo...RapID consists of this mutant BirA* and an RNA component that contains an RNA motif of interest. Any proteins...

... in advance Before validating your edit, it’s important to know a few things about your target cells and...goal is to generate a clonal cell line, so it’s important to verify that you won’t need to screen hundreds...method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR). This technique...trace files of WT and edited cell lines. Then decomposition analysis is performed yielding a graph of edits...assessment of genome editing by sequence trace decomposition. (2014) Nucleic Acids Research Volume 42, Issue...