Stellenbosch University

4 ARTICLES PUBLISHED IN JoVE

Biochemistry

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Dana M. Freund¹, Katherine A. Sammons², Nokwanda P. Makunga³, Jerry D. Cohen¹, Adrian D. Hegeman²

¹Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota, ²Department of Horticultural Science, Department of Plant and Microbial Biology, Microbial and Plant Genomics Institute, University of Minnesota, ³Department of Botany and Zoology, Stellenbosch University

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Cancer Research

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Shankari Nair¹, Samantha Cairncross², Xanthene Miles¹, Monique Engelbrecht³, Peter du Plessis¹, Julie Bolcaen¹, Randall Fisher¹, Roya Ndimba¹, Charnay Cunningham⁴, Wilner Martínez-López⁵, Christian Schunck⁶, Charlot Vandevoorde¹

¹Radiation Biophysics Division, Nuclear Medicine Department, iThemba LABS, ²Department of Biotechnology, University of the Western Cape, ³Department of Medical Biosciences, University of the Western Cape, ⁴Division of Medical Physiology, Department of Medicine and Health Sciences, Stellenbosch University, ⁵Biodosimetry Service, Instituto de Investigaciones Biológicas Clemente Estable and Academic Unit on Radiation Protection, Faculty of Medicine, University of the Republic, ⁶MetaSystems Hard & Software GmbH

This protocol presents the slide preparation and automated scoring of the γ-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Immunology and Infection

Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow

Jason D. Limberis^*1, Alina Nalyvayko^*1, Janré Steyn², Jennifer Williams², Melanie Grobbelaar², Robin M. Warren^*2, John Z. Metcalfe^*3

¹Division of Experimental Medicine, University of California, San Francisco, ²DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, ³Division of Pulmonary and Critical Care Medicine, Zuckerberg San Francisco General Hospital and Trauma Centre, University of California, San Francisco

This protocol shows bead-beating combined with DNA capture bead purification provides a fast and consistent method for extracting DNA from Mycobacterium tuberculosis samples, making it an effective choice for next-generation sequencing applications.

Genetics

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Jennifer Williams^*1, Janré Steyn^*1, Emilyn Costa Conceição^*1, Felicia Bernita Wells¹, Melanie Grobbelaar¹, Nabila Ismail¹, Yonas Ghebrekristos^1,2, Christoffel Johannes Opperman^1,2,3,4, Sarishna Singh^1,2, Jason Limberis⁵, Fahd Naufal⁵, Brendon Coenrad Mann¹, Rebecca E. Colman^6,7, Timothy Rodwell^*6,7, Robin Mark Warren^*1, John Z. Metcalfe^*5

¹Division of Molecular Biology and Human Genetics, SAMRC Centre for TB Research, DSI-NRF Centre of Excellence for Biomedical TB Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, ²National Health Laboratory Service, Green Point Tuberculosis Laboratory, ³Division of Medical Microbiology, Department of Pathology, University of Cape Town, ⁴Division of Medical Microbiology, Department of Pathology, University of Cape Town, ⁵Division of Pulmonary and Critical Care Medicine, Zuckerberg San Francisco General Hospital and Trauma Centre, University of California, San Francisco, ⁶Department of Medicine, University of California, San Diego, School of Medicine, ⁷Foundation for Innovative New Diagnostics (FIND), Campus Biotech

We present an optimized method for DNA extraction from decontaminated sputum using a matrix-based extraction method combined with a magnetic bead purification for downstream targeted next-generation sequencing of Mycobacterium tuberculosis.