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Practical 1: Safety in Practicals & DNA Isolation

Safety in Practicals
It is essential that you undertake an orientation training session before working in the biology laboratories.
This ensures that you are aware of all safety protocols, that you will know where to find equipment, and
will know how to keep the laboratory clean and functional. This will keep everyone safe.

In a group of four, make a tour of the laboratory and complete the questions below.

Working in a lab

Where are the washing facilities: for you,

and for equipment?

Where would you dispose of animal

material?

Where are the microscopes? Are they all the

same type?

Where are the clean microscope slides?

What happens to used slides?

How should you dispose of small pieces of

glass?

Where are the first aid materials?

Where should you keep your bags?

What should you do if you have long hair?

Why?
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Around your lab

What is personal protective equipment

(PPE)? Why is PPE necessary?

Why must feet be fully covered?

Why is there a NO eating and drinking rule

in the lab?

What do you do if there is a fire?

What is a risk assessment? Why should a

risk assessment be formulated before
experiments?

AFTER you have completed these questions and discussed safety issues, your demonstrator will check your
answers and sign off.

Demonstrator signature: ________________________________________________________________

Demonstrator name: ________________________________________________________________

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DNA Isolation

Exploration (what is this practical about)

“Understanding the molecular basis of inheritance has transformed how Biology is investigated forever.”
This statement from the popular science book: Life’s Greatest Secret by Matthew Cobb (a great read on
the story of the discovery of the genetic code) encapsulates the impact molecular biology has had on all
aspects of the study of Biology. Everything from forensic analysis, paternity testing, testing for genetically
modified (GM) crops, personalised medicine through to archaeological investigations into ancient tribe
migrations and the conservation of koalas and Tasmanian devils requires DNA sequence analysis.
Identifying the underlying mutations that cause cancers and hence determining potential treatments,
understanding the molecular basis of inflammation, diagnosing genetic mutations and investigating the
individual’s underlying genetic predisposition to diabetes, addictions and obesity are just some of the
research directions at Sydney University that rely on DNA sequencing. In all these examples the first step
is to isolate pure DNA that can be used as a template for downstream processes such as sequencing.
An organism’s genetic information is stored in this remarkable molecule, DNA, as a sequence of bases:
Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) which are attached chemically to a sugar-
phosphate backbone. Each cell of that organism (with a few exceptions) contains one complete copy of this
information as DNA and this is referred to as the genome. It is the organism’s unique identifier or
“barcode”. Not only does this information code for the proteins made by the organism but also the
regulation of their expression. It is the regulatory regions that are the focus of much of medical research.
In this practical you will isolate high molecular weight DNA which would then be suitable for cloning, PCR or
sequencing. In the next practical you will estimate the yield and purity of this DNA and digest this DNA with
a restriction enzyme to assess whether it is biologically “clean” and can be used as a template by an enzyme.
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Practical 1: Isolate pure DNA.

Practical 2: explore methods for quantifying DNA. Devise criteria for estimating the yield and purity of a
DNA preparation and apply these to assess your isolated DNA preparation. Digest DNA preparation with
a restriction enzyme and separate DNA fragments by gel electrophoresis to determine the size of the
DNA, RNA contamination and whether the DNA sample could be digested by an enzyme, is it “biologically
clean”?

Practical 1

Lyse E. coli cells

Remove RNA by digestion with RNase

Remove protein

Recover and resolubilise DNA

Learning Outcomes: Achieved By:

What you will learn
How to isolate high molecular weight DNA
from a selected source
The changes to the protocol required for
isolating DNA from different sources
The purpose of each step in the protocol
and be able to predict the outcome if the
step was omitted (troubleshooting)
How to assess the yield and purity of your
preparation by UV spectrophotometry and
to identify the extent of some possible
contaminants
How you will learn it
By doing it!
By comparing the differences in protocols designed to
isolate DNA from different sources
By reading over the experimental considerations and
observing the product after each step
By obtaining a suitable UV spectrum for your DNA
preparation (a spectrum with a peak in the working
range of the spectrophotometer) and interpreting it in
the light of principles explored in the next session.
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Before coming to this practical

• Login to Canvas and view any information for the practical.

• View the video on how to pipette
• Read through this practical thoroughly and complete the tasks below
• Review the lectures on nucleic acids.

Practical Activities

Safety Guidelines

Chemicals used in this practical:

The nuclei lysis buffer and protein precipitation buffer contain components which
may cause irritation to skin. Avoid skin contact.

Isopropanol – avoid contact with skin, ingestion or inhalation.

70% ethanol (C 2 H 6 O) – avoid contact with skin, ingestion and inhalation

Some tips when working with DNA:

Always wear gloves. The solution is not dangerous to you but you are dangerous to
the DNA! Your sweat contains nucleases (enzymes which degrade DNA and RNA) so
gloves must be worn to prevent these nasty nucleases from degrading your precious
DNA.
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Before beginning the investigation: Getting to know your pipette

Before starting the DNA isolation you will need to be familiar with the equipment you will be using,
specifically the pipettes. You will be using a range of pipettes designed to deliver very small volumes (2
µL to 1000 µL) very accurately and efficiently. To do this you need to be familiar with how to select the
appropriate pipette, set the volume and dispense the solution correctly. You will be tested on your pipette
skills
You must be familiar with the following dispensing tools:

• P20 pipette (range 2 – 20 µL)

• P200 pipette (range 20 – 200 µL)
• P1000 pipette (range 200 – 1000 µL or 0.2 - 1.0 mL)

For this part of the practical, work individually

Your demonstrator will show you how to set and use the pipettes found in the laboratory: P20, P200 and
P1000. There is a quick reference table in Appendix I with the volume range, tips and setting of each of
the pipettes commonly found in the laboratory, along with further information.

Follow the protocol below and complete the table:

1. Accurately dispense 1000 µL (1.0mL) of water into a microfuge tube, known commonly as an
Eppendorf tube. Add 10 µL of the P20 solution provided and mix well by vortexing.
2. Into a second Eppendorf tube add 900 µL water, followed by 100 µL of the P200 solution provided.
Mix well by vortexing.
3. Into a third Eppendorf tube add 750 µL water. Dispense 250 µL of the P1000 solution provided and
mix well by vortexing.
4. Spectrophotometers will have already been set-up in photometric mode at the desired wavelength.
Your demonstrator will briefly show you how to blank and measure the absorbance. Using a cuvette
filled with water, zero the spectrophotometer then obtain the absorbance for all three of your
solutions. Record your results below.
5. Your demonstrator will sign off on your results. If one of your measurements is not within the
acceptable range repeat the dilution and resubmit your absorbance to your demonstrator.
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1 mL water + 10 900 µL water + 750 µL water +

µL P20 coloured 100 µL P200 250 µL P1000
solution coloured solution coloured solution

Volume of Absorbance
Coloured Volume of
Pipette coloured 2nd attempt if
solution water (µL) 1st attempt
solution (µL) required

P20 P20 1000 10

P200 P200 900 100

P1000 P1000 750 250

Pipetting proficiency

Signed (demonstrator):
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Investigation: Isolation of DNA

The genetic material contained in an organism (the genome) is organized into chromosomes. Genes (functional
units of DNA, which is about the best definition of a gene!) are located within these chromosomes. Different
organisms have different numbers of chromosomes, depending, in part, on the complexity of the organism
and, obviously, on the amount of genetic material. (These don’t always equate). Each chromosome contains
a single molecule of DNA. This DNA molecule can be as long as 1 cm and has a very high molecular weight
(>1010).
The total DNA contained in a single human nucleus (as 46 separate double stranded molecules) is over 2 m
in length. DNA this length obviously doesn’t just ‘float around’ in the cell, but is packaged into compact
chromosomes. This packaging is achieved by specific proteins. In eukaryotes these proteins are histones; small
positively charged molecules called histones that interact with the negatively charged phosphates of the
DNA.
Bacteria have much less genetic material than higher eukaryotic organisms, but this too must be packaged
into more compact structures to fit into the cell. E. coli contains ~4 million base pairs of DNA packaged in a
single supercoiled circle of DNA about 1300 µm in length. As the bacterium is between 1 and 3 µm in
length this DNA must be folded within the cell. Electron micrographs of the E. coli chromosome show multiple
loops of DNA radiating from a central, protein dense ‘scaffold’ region.

Your task in this investigation:

In this practical you will isolate high molecular weight DNA and then in the following week estimate its yield
and purity.

What we expect of you:

• Work diligently and in line with the safety guidelines for this practical
• Carefully watch the demonstration before undertaking your own experiment
• Accurately record your results
Don’t worry about mistakes. We expect this will be a process of trial and errors. You should learn from
your mistakes.

How you are going to do this:

For this investigation, work in pairs.

You will follow the methods below to conduct the following sequence:
A. Lyse (split open) the cells
B. Digest the RNA with RNase
C. Remove the protein
D. Recover the nucleic acid
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For this investigation you will be provided with the following equipment:

• Biological source: E. coli suspension

• Nuclei Lysis Buffer
• 80oC water bath
• Ribonuclease (RNase)
• Protein Precipitation Solution
• Bench centrifuges capable of spinning “Eppendorf” tubes at 12,000 × g
• 1.5 mL “Eppendorf” tubes
• Esky with ice
• Isopropanol (room temperature)
• 70% (v/v) ethanol (room temperature)
• TE (10 mM Tris 1 mM EDTA,pH 8) known as Rehydration Solution
• P20, P200 and P1000 pipettes with tips
• Plastic Pasteur pipettes

Methods
Technical Notes: Experimental considerations

• Any procedure designed to isolate pure, intact, high molecular weight DNA will need to consider
the ‘natural’ packaged state of the DNA inside the cell. For this reason most DNA isolation
methods from bacterial, yeast or animal cells involve cell lysis, either with detergent or enzymes
or some combination of the two. Isolation of DNA from plant cells has to be more rigorous due
to the tough cellulose cell wall. Grinding in liquid N 2 is a common way of achieving cell lysis with
plant cells.
• The RNA is removed with ribonuclease. As RNA and DNA have very similar structures it is
difficult to select a chemical method that can distinguish the two nucleic acid polymers and only
degrade one while keeping the other intact. Enzymes, however are very specific and
ribonuclease will only digest RNA, not DNA.
• This is followed by removal and denaturation of the associated proteins of the scaffold. This is
usually achieved using high ionic strength and organic solvents (phenol and chloroform).
Proteolytic enzymes e.g. proteinase K are often also employed to specifically and gently
remove and digest the protein. This kit uses a protein precipitation solution (a proprietary
secret)
• The remaining DNA is precipitated in isopropanol and the pellet washed with 70% (v/v)
ethanol. Nucleic acid polymers, DNA and RNA, are insoluble in ethanol concentrations of >66%.
This precipitation is greatly aided by the inclusion of cations (e.g. Na+). What is the role of these
cations in the precipitation process?
• The DNA precipitate is then resolubilised in a buffer with EDTA, a chelator of divalent cations
such as Mg2+. Throughout the whole procedure, note what steps are taken to minimize the action of
endogenous and exogenous DNases.
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A. Cell Lysis
Each pair will be provided with 1 mL of an E. coli suspension (dissolved in 0.1 M NaCl, 10 mM EDTA,
pH 8) in a 1.5 mL “Eppendorf” centrifuge tube.

1. Centrifuge for 2 min at 12,000 × g. Remove and discard the supernatant (the liquid)
into the beaker labeled waste. This pellets the cells containing the DNA.
2. Add 600 µL Nuclei Lysis Buffer. You will need to use the P1000 pipette with the blue
tip. Gently pipette up and down until the cells are resuspended. This may take some
time.
3. Incubate for 5 min in the 80oC waterbath to lyse the cells then allow to cool to room
temperature.
Step 1
Centrifuge Gently
Step 2
for 2 min at resuspend
600 µL cells with a
12,000 × g.
Nuclei Lysis pipette
Discard the
supernatant Buffer

Pellet
containing
DNA

Step 3
Incubate cell suspension for
5 min at 80oC to lyse cells.
Cool to room temperature.

B. Digesting RNA.
Cells contain a lot of RNA (approximately 5 × more than DNA. It is very similar in structure and has to be
removed with a specific enzyme, Ribonuclease (RNase) that only degrades RNA (not DNA))

4. Add 3 µL RNase (use a P20 pipette with a yellow tip for this addition). Invert the tube
2 – 5 times to mix.
5. Incubate at 37oC for 30 min. Allow sample to cool to room temperature.

Step 5
Step 4
Incubate cell suspension
3 µL RNase for 30 min at 37oC to
digest RNA. Cool to
room temperature.
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C. Precipitating the Protein

6. Add 200 µL Protein Precipitation Solution to the RNase-treated solution. Vortex
vigorously at high speed for 20 sec to mix.
7. Incubate the sample on ice for 5 min
8. Centrifuge at 12,000 × g for 3 min.

Step 7
Incubate for 5 min on
ice to precipitate the
Step 6 protein.

200 µL Protein
Precipitation Step 8
Solution. Vortex
20 sec Centrifuge for 3
min at 12,000 × g.

9. Transfer the supernatant containing the DNA to a clean 1.5 mL Eppendorf tube
containing 600 µL isopropanol (room temperature). Use a P200 with a yellow tip to
transfer the supernatant. Leave the last bit of liquid on top of the protein pellet. It is
better to collect less liquid than to contaminate your precious DNA with some of the
protein pellet.
10. Gently invert and thread-like strands of DNA should start to form.

Supernatant Step 10
containing Gently invert and
DNA…KEEEP!!!! watch for the DNA
Supernatant threads to appear.
containing
DNA…and
Pellet containing isopropanol
protein DISCARD
Step 9
Carefully transfer the
supernatant to a
clean tube containing
600 µL isopropanol.
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D. Recovering your DNA

11. Centrifuge at 12,000 × g for 2 min.

12. Pour off the supernatant carefully and drain the pellet (containing the DNA) on paper
towel for at least 5 min.
13. Add 600 µL 70% (v/v) ethanol and gently invert the tube several times to wash
residual salts from the pellet.
14. Centrifuge at 12,000 × g for 2 min. Carefully remove the ethanol with a P200
pipette.
15. Drain tube on paper towel then air dry for ~10 min
16. Add 100 µL Rehydration Solution (10 mM Tris 1 mM EDTA, pH 7.4). The tube must then
be labeled with your name as it will be stored in the cold room (~5oC) until the next
practical session.

Hand your tube containing your precious DNA to your demonstrator

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Results

Take images of your tube at the steps marked with the photo icon.

Consolidation (what did I learn)

Your demonstrator will guide a group discussion using the questions below:

• What are the structural differences between RNA and DNA?

• What steps in the procedure above are designed to distinguish between DNA and RNA?

• How would you change the protocol to isolate DNA from other sources such as plants or blood?

• What is the purpose of washing the pellet in 70% ethanol?

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Assessment

Concepts and skills from this practical may be used in the Information Transfer module quizzes and in the
final exam.

Reflections

In this practical you have learnt about DNA isolation. We would like to assess how well you think you have
done (there is no right or wrong answer). Please tick a box against each statement. Once complete ask
your demonstrator to sign off.

Strongly Agree Neutral Disagree Strongly

agree disagree
I understand the
process of DNA
isolation and why
protocols are done in
an order
I can isolate DNA
I am able to identify
the likely contaminants
of DNA isolation
There was sufficient
time to complete the
practical
The practical notes
were good
Class discussions aided
my understanding

Any additional comments:

Connections to lectures, set texts, online elements to unit

This practical complements, and is sequenced with lectures on nucleic acids in this Unit of study. Please
refer to the Information Transfer Module on Canvas to view all related materials and Appendices I to V.