Pulsed-field gel electrophoresis of budding yeast chromosomes.
Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along with details of embedding cells in agarose before removal of cell walls. We then detail the procedure to remove protein and RNA from chromosomes and how naked chromosomes are loaded into agarose gels before being subjected to electrophoresis. Finally, we describe how the separated chromosomes can be visualized and photographed.
Maringele
Laura
L
University of Newcastle, School of Clinical Medical Sciences--Newcastle General Hospital, Newcastle upon Tyne, UK.
Lydall
David
D
D002458:Cell Fractionation / Q000379:methods; D015825:Chromosomes, Fungal / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D009172:Mycology / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000254:growth & development; D012685:Sepharose; D013194:Staining and Labeling
D012685:Sepharose; D004262:DNA Restriction Enzymes