Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 7

Laboratory 4c Yeast DNA Extraction Protocol optimized by Prithanjan Bhattacharya and Kyle Siaotong, Biotechnology students, 2010 Background Yeast is the common name of a group of unicellular fungi. A variety of different types of yeasts are used in brewing and baking. Baker’s yeast (Saccharomyces cerevisiae) is used at home and in industry as a leavening agent for bread. Yeast cells are also grown in liquid culture and used as production molecules for some biotechnology products. As a production organism, yeast is well studied and its DNA has been sequenced. Yeast DNA can be extracted from yeast cells fairly easily. For the extraction, the yeast cells must be rehydrated and incubated at 37°C in a broth solution of glucose and dH2O. In this environment, a large density of cells will be available for DNA extraction. Purpose Can DNA be extracted from yeast cells grown in broth culture? Materials Balance, analytical Balance, tabletop milligram Weigh paper, 7.6×7.6 cm Weigh boat, 3.5”×3.5” Lab scoops Glucose dH2O Media bottles, 125 mL Shaking incubator, 37°C TRIS-HCl EDTA, disodium salt Sodium chloride Triton X-100 SDS, 10% Tubes, 15 mL, capped Tube racks for 15-mL tubes TRIS (TRIS base) Glass stirring rod pH meter or pH paper 2-mL centrifuge tubes High-speed microcentrifuge 95% ethanol (EtOH) Lab tissues Hairdryer Heat block Fleischmann’s RapidRise® yeast or other baker’s yeast Tabletop centrifuge for 15-mL conical tubes Protease (papain or Adolph’s® meat tenderizer) Micropipets and tips, P-1000 and P-100 Procedure Modified from procedures originally published by Hoffman and Winston, Gene, 1987. Pre-Lab: Preparation of Yeast Broth Culture and Solutions (for a class of 16 lab groups) 1. At least 36 hours prior to DNA extraction, prepare five yeast broth cultures, each with 8 g baker’s yeast, 0.2 g glucose, and 37°C dH2O added up to the 40-mL graduation. Transfer the mixtures to 125-mL media bottles. Incubate for 36 hours at 37°C in a shaking incubator (250 rpm), until the broth is very cloudy and bubbly. Store at 4°C until ready to use. 2. Prepare 50 mL of Cell Lysis Solution that contains: 5 mL 1M NaCl 5 mL 100mM Tris-HCl (pH 8.0) 5 mL 10mM EDTA (pH 8.0) 1 mL 2% Triton X-100 50 µL of 10% SDS Add dH2O until a total of 50 mL is reached. Gently mix without allowing it to get foamy. Store at room temperature. 3. Prepare 5 mL of 5% protease solution (5% papain or Adolph’s® meat tenderizer). Store at 4°C. 4. Prepare 50 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Store at 4°C. 5. Prepare 5 mL of 4M ammonium acetate solution. Store at room temperature. 72 Chapter 4   Laboratory Manual