JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION

DOI: https://doi.org/10.1128/jmbe.v18i2.1348

Supplemental Materials
for
Bacterial DNA Extraction Using Individual Enzymes
and Phenol/Chloroform Separation
Mitchell Henry Wright1*, Joseph Adelskov2, and Anthony Carlson Greene2
1
Division of Environmental and Biomolecular Systems, Institute of Environmental
Health, Oregon Health & Science University, Portland, OR 97239-3098,
2
School of Natural Sciences, Griffith University, Nathan Campus, Queensland, Australia

Table of Contents
(Total pages 7)

Appendix 1: Pre-laboratory questions

Appendix 2: Pre-laboratory answer key
Appendix 3: DNA extraction flowchart
Appendix 4: Agarose gel preparation for electrophoresis

*Corresponding author. Mailing address: Division of Environmental

and Biomolecular Systems, Institute of Environmental Health,
Oregon Health & Science University, 3181 S.W. Sam Jackson
Park Rd., Portland, OR, 97239-3098. Phone: 503-346-3434.
E-mail: wrigmit@ohsu.edu.
Received: 2 May 2017, Accepted: 3 July 2017, Published:
1 September 2017.
©2017 Author(s). Published by the American Society for Microbiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-
Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode),
which grants the public the nonexclusive right to copy, distribute, or display the published work.

JMBE ▪ 1752 N Street NW, Washington, DC 20036

(202) 942-9299 ▪ fax (202) 942-9329 ▪ jmbe@asmusa.org
http://asmscience.org/jmbe
Appendix 1: Pre-laboratory questions.

Question 1) What are the roles of the following reagents in DNA extraction?

Proteinase K: _______________________________________________________________

RNase A: _______________________________________________________________

SDS: _______________________________________________________________

Question 2) What are the roles of achromopeptidase and lysozyme? How do they differ?

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

Question 3) What is the purpose of the Phenol:Chloroform:Isoamyl alcohol (PCI) step?

Why is it necessary to invert and then centrifuge this mixture?

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

1
Question 4) Why is it important to run a ladder standard alongside samples when

performing agarose gel electrophoresis? What are the two types of information obtainable

from using a ladder?

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

2
Appendix 2: Pre-laboratory answer key.

Question 1) What are the roles of the following reagents in DNA extraction?

Proteinase K: Broad action proteinase that degrades protein released after cell lysis

RNase A: Degrades ribonucleic acid in the cell lysate

SDS: Disrupts the cell membrane causing cell lysis

Question 2) What are the roles of achromopeptidase and lysozyme? How do they differ?

Achromopeptidase and lysozyme degrade the bacterial cell wall. Lysozyme can cleave the bond

between the n-acetyl-glucuronic acid and n-acetyl-muramic acid monomers of the peptidoglycan

chains. Achromopeptidase cleaves the tetrapeptide linkages between peptidoglycan chains.

Question 3) What is the purpose of the Phenol:Chloroform:Isoamyl alcohol (PCI) step?

Why is it necessary to invert and then centrifuge this mixture?

PCI organic phase separates DNA from cell debris and protein in the cell lysate. Invert mixing

the solution allows cell debris to transfer to the organic phase without damaging the DNA.

Centrifugation of the mixture forces remaining protein and cell debris precipitated in the aqueous

phase to migrate to the organic:aqueous interface.

3
Question 4) Why is it important to run a ladder standard alongside samples when

performing agarose gel electrophoresis? What are the two types of information obtainable

from using a ladder?

A ladder standard is run alongside samples to act as a reference. The ladder has bands with

known lengths and relative concentration to allow estimation of sample DNA fragment size and

concentration.

4
Appendix 3: DNA extraction flowchart.

5
Appendix 4: Agarose gel preparation for electrophoresis.

A. Materials:

a. Molecular Biology Grade Agarose (Fisher Scientific)

b. 1×TAE Running Buffer

c. GelRed™ (Biotium)

d. λHind III/DNA Ladder (TOOLS Life Sciences)

e. Loading Dye

B. Agarose Gel Preparation and Electrophoresis:

a. Prepare a 1.2% agarose gel: 0.6 g agarose, 50 mL 1× TAE buffer

b. Boil for 1 minute and allow to cool to ~55°C

c. Add 2µL of GelRed™ (Biotium)

d. Mix by swirling and pour into prepared casting rig

e. Allow gel to set (until gel turns opaque, ~15 minutes)

f. Place gel in electrophoresis apparatus and submerge with 1×TAE running buffer

g. Prepare samples by mixing loading dye with DNA sample on a strip of paraffin: 2

µL loading dye with 5µL DNA sample

h. Load samples and λHind III linear standards into gel wells

i. Electrophoresis at 120V, 80 mA, for 40 minutes

j. Visualize bands under U.V. light