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