Current Protocols Essential Laboratory Techniques最新文献

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR/Cas) are revolutionary tools for predictable and repeatable genome editing. In this article, the CRISPR/Cas9 system will be used to engineer the genome of the yeast Saccharomyces cerevisiae. As a model organism utilized across biological disciplines, efficient and tailorable methods for engineering the yeast genome are indispensable for a variety of research, educational, and commercial applications. The protocols described here incorporate a simple restriction-free cloning strategy and digestion-based screening method that can be readily and easily modified to suit user-designed experimental needs. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Recombinant pCAS plasmid preparation

Basic Protocol 2: Barcode/editing fragment assembly

Basic Protocol 3: Gene editing by yeast co-transformation

Alternate Protocol: Competent yeast preparation and transformation by a lithium acetate/single-stranded carrier method

Stored biological materials should have minimal pre-analytical variations in order to provide researchers with high-quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK-8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors.

Basic Protocol: Flash-freezing fresh tissue with aluminum platform

Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen

Alternate Protocol 2: Freezing fresh tissue with dry ice

Conferences play an important role in enabling trainees to develop and apply competencies in science, in communication, and in networking during biomedical PhD and postdoctoral training. This article offers guidelines for trainees on how to use conferences to initiate, sustain, and strengthen connections, including in virtual conference formats which could become the norm in the future. Additionally, it provides tips for expanding professional networks via broad mechanisms such as informational interviews. Recommendations in this manuscript are applicable to trainees pursuing diverse career paths in different STEM fields including education, scientific research, policy, advocacy, consulting, and communication. © 2020 Wiley Periodicals LLC.

In order for the scientific enterprise to ensure equitable participation for all identities, the settings of professional research labs must cultivate an environment that is inclusive of all backgrounds. We explore here strategies to consider for research labs interested in cultivating inclusive environments. Investigators enacting inclusive strategies must understand the social context of the lab members and their reasons for engaging in science research. For this to be authentic, principal investigators should spend time exploring their own social positioning as well as the purpose of their professional engagement. We unpack the philosophies behind these constructs and provide specific suggestions to prepare individuals to fully engage in the practice of inclusive mentoring in science research labs. © 2020 Wiley Periodicals LLC.

The laboratory notebook, whether in hardcopy or electronic (ELN) format, represents a true, chronological record of a scientist's bench work, incorporating the primary source of all materials and information related to the design, execution, and outcomes (data) for a specific experiment. The notebook content thus represents a precise, legible record of what was done, why, and by whom. It also describes the outcome of the experiment and how this is related to the initial hypothesis on which performing the experiment was based. As an archive, a laboratory notebook allows others previously unassociated with the work to understand what was done and, if necessary, independently recreate the experiments. A laboratory notebook and its associated records (e.g., computer printouts or HPLC traces) also represent the vital record of the conception date of an invention and its reduction to practice, providing a timely and necessary legal record to support and defend patent applications. © 2020 Wiley Periodicals LLC.

This article provides an overview of foundational concepts of business strategy and business development that scientists can apply to starting and expanding their research programs. It covers topics including: defining a value proposition, identifying stakeholders, considering research gaps, strategic collaborations, responsible hiring, strategic planning, and time management. © 2020 by John Wiley & Sons, Inc.

For success in research careers, scientists must be able to communicate their research questions, findings, and significance to both expert and nonexpert audiences. Scientists commonly disseminate their research using specialized communication products such as research articles, grant proposals, poster presentations, and scientific talks. The style and content of these communication products differ from the language usage of the general public and can be difficult for nonexperts to follow and access. For this reason, it is important to tailor scientific communications to the intended audience in order to ensure that the communication product achieves its goals, especially when communicating with nonexpert audiences. This article presents a framework to increase access to research and science literacy. It addresses aspects of communication that scientists should consider when producing a scientific communication product: audience, purpose, format, and significance (research narrative). These factors are essential for understanding the communication scenario and goals, which provide guidance when tailoring research communications to different audiences. © 2020 by John Wiley & Sons, Inc.

Cover: In Meneely et al. (https://doi.org/10.1002/cpet.35), Hermaphrodites (A) and males (B). Note that gonads have different shapes and that males have an elaborate fan-shaped tail. Images copyrighted by WormAtlas.org and used by permission.

Arabidopsis thaliana is a small plant of significant economic and agronomic importance. While Arabidopsis is not a crop plant, it is not so different in its fundamental properties. Arabidopsis is a member of the Brassicaceae family, which constitutes one of the world's most economically important plant groups. According to the United Nations, globally Brassicaceae crops are worth $31 billion, and the number is likely to increase since a number of related species within this family are underutilized edible varieties. Its small size is an advantage to researchers with limited space and funding; simply put, a smaller plant requires fewer resources. Arabidopsis has been studied most intensely for the last 40 years and officially became a model plant in the late 1990s. Since then, the community has developed genetic and genomic resources so numerous that the barrier to entry to studying Arabidopsis is relatively low. This article provides a primer to how Arabidopsis came to be a model organism and highlights essential techniques every Arabidopsis researcher should be aware of to advance the pace of discovery. © 2019 by John Wiley & Sons, Inc.

In science and engineering, there are a range of easy-to-access software applications for smart phones, tablets, and laptop and desktop computers that greatly simplify basic and advanced laboratory research activities. The available software programs include numerous mobile and web-friendly applications for electronic laboratory notebooks, project planning, solution calculations, and advanced scientific calculations and analysis, and even simple-to-use apps for your favorite scientific meetings. A typical collection of mobile-friendly applications is discussed here in the context of preparing, performing, and presenting your research. © 2019 by John Wiley & Sons, Inc.