Current Protocols Essential Laboratory Techniques最新文献

CRISPR/Cas9-Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction-Free Cloning and a Rapid BamHI Digest Readout 基于CRISPR/Cas9的酿酒酵母ADE2基因的基因组编辑，无限制性克隆和快速BamHI文摘读取

Current Protocols Essential Laboratory Techniques Pub Date : 2020-12-09 DOI: 10.1002/cpet.45

Allison R. Sirois, Nils Pilotte, Steven A. Williams, Lori J. Saunders

{"title":"CRISPR/Cas9-Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction-Free Cloning and a Rapid BamHI Digest Readout","authors":"Allison R. Sirois, Nils Pilotte, Steven A. Williams, Lori J. Saunders","doi":"10.1002/cpet.45","DOIUrl":"10.1002/cpet.45","url":null,"abstract":"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 preparationBasic Protocol 2: Barcode/editing fragment assemblyBasic Protocol 3: Gene editing by yeast co-transformationAlternate Protocol: Competent yeast preparation and transformation by a lithium acetate/single-stranded carrier method","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.45","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42942568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Validation of a Novel, Flash-Freezing Method: Aluminum Platform 一种新型快速冷冻方法的验证:铝平台

Current Protocols Essential Laboratory Techniques Pub Date : 2020-11-25 DOI: 10.1002/cpet.46

Ahmet Imrali, Christine S. Hughes, Abigail S. Coetzee, Francesca R. Delvecchio, Amina Saad, Rhiannon Roberts, Claude Chelala, Jo-Anne ChinAleong, Hemant M. Kocher

{"title":"Validation of a Novel, Flash-Freezing Method: Aluminum Platform","authors":"Ahmet Imrali, Christine S. Hughes, Abigail S. Coetzee, Francesca R. Delvecchio, Amina Saad, Rhiannon Roberts, Claude Chelala, Jo-Anne ChinAleong, Hemant M. Kocher","doi":"10.1002/cpet.46","DOIUrl":"10.1002/cpet.46","url":null,"abstract":"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 platformAlternate Protocol 1: Freezing fresh tissue with liquid nitrogenAlternate Protocol 2: Freezing fresh tissue with dry ice","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.46","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39114621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

The Importance of Conferences and Networking in PhD and Postdoctoral Training 会议和网络在博士和博士后培养中的重要性

Current Protocols Essential Laboratory Techniques Pub Date : 2020-10-08 DOI: 10.1002/cpet.44

Shoba Subramanian

引用次数: 1

Reflections and Actions for Creating an Inclusive Research Environment 创造包容性研究环境的思考与行动

Current Protocols Essential Laboratory Techniques Pub Date : 2020-10-08 DOI: 10.1002/cpet.43

Bryan Dewsbury, Shannon Seidel

引用次数: 1

Laboratory Notebooks and Data Storage 实验室笔记本和数据存储

Current Protocols Essential Laboratory Techniques Pub Date : 2020-06-19 DOI: 10.1002/cpet.42

Michael Williams, Donna Bozyczko-Coyne, Bruce Dorsey, Scott Larsen, Chris Powers, Sean Gallagher

引用次数: 0

Applying Business Strategies to Establish Your Research Program 运用商业策略建立你的研究计划

Current Protocols Essential Laboratory Techniques Pub Date : 2020-02-21 DOI: 10.1002/cpet.41

Thi A. Nguyen, Andrew D. Nguyen

引用次数: 0

Tailoring Scientific Communications for Audience and Research Narrative 为受众和研究叙事量身定制科学传播

Current Protocols Essential Laboratory Techniques Pub Date : 2020-01-24 DOI: 10.1002/cpet.40

Jessica A. Hutchins

{"title":"Tailoring Scientific Communications for Audience and Research Narrative","authors":"Jessica A. Hutchins","doi":"10.1002/cpet.40","DOIUrl":"10.1002/cpet.40","url":null,"abstract":"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.","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38504776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Issue Information TOC 发布信息TOC

Current Protocols Essential Laboratory Techniques Pub Date : 2019-12-19 DOI: 10.1002/cpet.30

引用次数: 0

Arabidopsis Thaliana: From Weed to Model Organism 拟南芥:从杂草到模式生物

Current Protocols Essential Laboratory Techniques Pub Date : 2019-12-02 DOI: 10.1002/cpet.38

Michael Diaz

{"title":"Arabidopsis Thaliana: From Weed to Model Organism","authors":"Michael Diaz","doi":"10.1002/cpet.38","DOIUrl":"10.1002/cpet.38","url":null,"abstract":"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.","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48605962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

The Use of Mobile Applications in Research 移动应用程序在研究中的使用

Current Protocols Essential Laboratory Techniques Pub Date : 2019-12-02 DOI: 10.1002/cpet.39

Sean R. Gallagher

引用次数: 1