CHAPTER TWELVE

Preparation of Genomic DNA fromSaccharomyces cerevisiaeJessica S. Dymond1The High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns HopkinsUniversity School of Medicine, Baltimore, MD, USA1Corresponding author: e-mail address: jsiege16@jhmi.edu

Contents

1.

MetISShttp

Theory

hods in Enzymology, Volume 529 # 2013 Elsevier Inc.N 0076-6879 All rights reserved.://dx.doi.org/10.1016/B978-0-12-418687-3.00012-4

154

2. Equipment 154 3. Materials 154

3.1

Solutions & buffers 154 4. Protocol 155

4.1

Preparation 155 4.2 Duration 155

5.

Step 1 Harvesting Cells from the Overnight Culture 155 5.1 Overview 155 5.2 Duration 155 5.3 Tip 156

6.

Step 2 Initial DNA Extraction 156 6.1 Overview 156 6.2 Duration 157 6.3 Tip 157 6.4 Tip 157 6.5 Tip 157 6.6 Tip 157

7.

Step 3 Purification of the Crude DNA Preparation 157 7.1 Overview 157 7.2 Duration 158 7.3 Tip 159 7.4 Tip 159

References

160 Source References 160

Abstract

The ability to isolate genomic DNA rapidly and effectively for analysis by PCR, Southernblotting, or other methods is an essential skill. This protocol provides a fast and efficientmethod for obtaining genomic DNA from S. cerevisiae.

153

154 Jessica S. Dymond

1. THEORY

This method to extract genomic DNA from yeast is fast and reliably

generates ample DNA for multiple applications. If high molecular weight

genomic DNA is required, an alternative method employing enzymatic

lysis, rather than mechanical lysis, should be employed (Boeke et al., 1985).

2. EQUIPMENT

Refrigerated centrifuge

Refrigerated microcentrifuge

Vortex mixer

Micropipettors

Micropipettor tips

15-ml polypropylene centrifuge tubes

1.5-ml microcentrifuge tubes

Glass beads, 425–600 mm, acid-washed

3. MATERIALS

YPD media

Tris base

EDTA

Hydrochloric acid (HCl)

Sodium chloride (NaCl)

Triton X-100

Sodium dodecyl sulfate (SDS)

Phenol/chloroform/isoamyl alcohol, 25:24:1 (v/v/v)

Ethanol

Ammonium acetate (NH4OAc)

RNase A

Sterile H2O

3.1. Solutions & buffersStep 1 Lysis Buffer

Component

Final Concentration Stock Amount

Tris–HCl, pH 8.0

10 mM 1 M 10 ml

EDTA, pH 8.0

1 mM 0.5 M 2 ml

155Preparation of Genomic DNA from Saccharomyces cerevisiae

NaCl

100 mM 5 M 20 ml

Triton X-100

2% (v/v) 100% 20 ml

Sodium dodecyl sulfate (SDS)

1% (v/v) 10% (w/v) 100 ml

Add water to 1 l

TE Buffer

Component

Final concentration Stock Amount

Tris–HCl, pH 8.0

10 mM 1 M 10 ml

EDTA, pH 8.0

1 mM 0.5 M 2 ml

Add water to 1 l

4. PROTOCOL

4.1. Preparation

Inoculate a single yeast colony into 10 ml YPD and grow, shaking, at

30 �C overnight.

4.2. Duration

Preparation

1 day

Protocol

3–4 h

See Fig. 12.1 for the flowchart of the complete protocol.

5. STEP 1 HARVESTING CELLS FROM THEOVERNIGHT CULTURE

5.1. Overview

Isolate yeast cells from the overnight culture and remove trace media.

5.2. Duration15 min

1.1 Transfer the overnight culture into a 15-ml centrifuge tube.

1.2 Centrifuge at 3600 rpm at 4 �C for 5 min.

1.3 Resuspend the cell pellet in 1 ml H2O. Transfer cells to a 1.5-ml

microcentrifuge tube.

1.4 Centrifuge at 3600 rpm at 4 �C for 5 min. Pour off the supernatant.

Figure 12.1 Flowchart of the complete protocol, including preparation.

Figure 12.2 Flowchart of Step 1.

156 Jessica S. Dymond

5.3. TipThe protocol may be temporarily halted at this point; store the cell pellet at

�20 �C overnight.

See Fig. 12.2 for the flowchart of Step 1.

6. STEP 2 INITIAL DNA EXTRACTION

6.1. Overview

Cells are lysed and a crude DNA preparation is extracted.

157Preparation of Genomic DNA from Saccharomyces cerevisiae

6.2. Duration35–45 min

2.1 Resuspend the cell pellet in 200 ml Lysis Buffer.2.2 Add 200 ml glass beads and 400 ml phenol/chloroform/isoamyl alco-

hol (25:24:1).

2.3 Vortex for 2 min (single tube vortexer) or 10 min (multitube

vortexer).

2.4 Add 400 ml TE Buffer and mix by vortexing briefly.

2.5 Centrifuge 10 min at maximum speed at room temperature.

2.6 Transfer 400 ml of the aqueous layer to a new microcentrifuge tube.

2.7 Add 1 ml ice-cold ethanol. Mix by inverting.

2.8 Centrifuge for 5 min at maximum speed at room temperature.

2.9 Wash the pellet with 70% ethanol.

2.10 Dry the pellet at room temperature for �5 min.

2.11 Resuspend the pellet in 500 ml TE Buffer.

6.3. TipRemove glass beads from the top of the tube. Incomplete closure of themicrocentrifuge tube

will cause phenol/chloroform contamination of equipment and dissolution of tube labels.

6.4. TipCell lysis can be confirmed by examining the resulting cell slurry under a microscope. If

lysis is incomplete, continue vortexing in short intervals until lysis is nearly complete.

6.5. TipThis preparation may be used for robust PCR applications, although the DNA will

not be stable for long periods of time. For extremely stable genomic DNA, complete

Step 3.

6.6. TipThe protocol may be temporarily halted here. Store the pellet in TE at 4 �C overnight.

See Fig. 12.3 for the flowchart of Step 2.

7. STEP 3 PURIFICATION OF THE CRUDE DNAPREPARATION

7.1. Overview

Contaminating RNA and cellular debris are removed.

Figure 12.3 Flowchart of Step 2.

158 Jessica S. Dymond

7.2. Duration1–2 h

3.1 Add 15 ml 2 mg ml�1 RNase A.

3.2 Incubate at 37 �C for 30 min.

3.3 Add 500 ml phenol/chloroform/isoamyl alcohol (25:24:1). Vortex for

30 s (single tube vortexer) or 5 min (multitube vortexer) to mix.

3.4 Centrifuge for 5 min at maximum speed at room temperature.

3.5 Transfer 400 ml of the aqueous layer to a clean microcentrifuge tube.

3.6 Add 1 ml ice-cold ethanol, and 10 ml 4 M ammonium acetate. Invert

to mix.

3.7 Centrifuge for 10 min at maximum speed at room temperature.

3.8 Wash the cell pellet with 70% ethanol.

159Preparation of Genomic DNA from Saccharomyces cerevisiae

3.9 Dry the pellet at room temperature for �5 min.

3.10 Resuspend the pellet in 100 ml TE Buffer.

7.3. TipIf a very high yield of genomic DNA is required, incubate at�20 �C for at least 1 h

in Step 3.6.

7.4. TipIf a very high yield of genomic DNA is required, perform the centrifugation for Step

3.7 at 4 �C.See Fig. 12.4 for the flowchart of Step 3.

Figure 12.4 Flowchart of Step 3.

160 Jessica S. Dymond

REFERENCESReferenced LiteratureBoeke, J. D., Garfinkel, D. J., Styles, C. A., & Fink, G. R. (1985). Ty elements transpose

through an RNA intermediate. Cell, 40, 491–500.

SOURCE REFERENCESHoffman, C. S. (1997). Preparation of yeast DNA. Current Protocols in Molecular Biology, 39,

13.11.1–13.11.4.