Qingdao Agricultural University 2 articles published in JoVE Biology Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp Lina Qiu1, Daqing Huang1, Yongzhang Wang1, Haiyong Qu1 1College of Horticulture, Qingdao Agricultural University Isolated protoplasts of apple pulp cells were loaded with a calcium fluorescent reagent to detect cytoplasmic Ca2+ concentration. Bioengineering Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System Hegang Li*1, Huaiyuan Qin*1, Ning Zhang*1, Jinshan Zhao1, Jingjing Xin1, Flor M. Perez-Campo2, Huawei Liu1 1Qingdao Agricultural University, Qingdao, China, 2University of Cantabria, Santander y Torrelavega, Spain Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.