Techniques in Molecular Biology:
Introduction to DNA electrophoresis
A practical program
What is Molecular Biology?
“The branch of biology that deals with the structure
and function of the macromolecules essential to
life.”
Key areas of study:
– DNA
– RNA
– Proteins
– Lipids
– Glycans
– Small Molecules
Why is Molecular Biology important?
Understanding the molecular level helps us
understand how organisms function. This
potentially allows us to:
• Understand relationships between organisms
(phylogeny)
• Understand disease and potential treatment
• Develop new products – e.g. insulin,
antibiotics
How this program maps to the Syllabus
Content
Outcomes
ACSSU184
The transmission of heritable characteristics from one generation to the next involves DNA
and genes
ACSHE120
ACSHE135
ACSIS199
Science and technology contribute to finding solutions to a range of contemporary issues;
these solutions may impact on other areas of society and involve ethical considerations
ACSHE160
People can use scientific knowledge to evaluate whether they should accept claims,
explanations or predictions
ACSIS125
Collaboratively and individually plan and conduct a range of investigation types, including
fieldwork and experiments, ensuring safety and ethical guidelines are followed
ACSIS132 ACSIS234
Use scientific knowledge and findings from investigations to evaluate claims
ACSIS145 ACSIS170
Summarise data, from students’ own investigations and secondary sources, and use
scientific understanding to identify relationships and draw conclusions
ACSIS204
Use knowledge of scientific concepts to draw conclusions that are consistent
with evidence
Stage 6 Biotechnology
9.6.5
Modern biotechnology includes recombinant DNA technology
Stage 6 Biotechnology
9.6.6
There are many applications and areas of research in biotechnology
How this program maps to the Syllabus
NSW Syllabus
Outcomes
Stage 5 LW3
Advances in scientific understanding often rely on
developments in technology, and technological advances
are often linked to scientific discoveries. Students identify
that during reproduction the transmission of heritable
characteristics from one generation to the next involves
DNA and genes.
SC4-17CW
Students explain how scientific understanding of, and
discoveries about the properties of elements, compounds
and mixtures relate to their uses in everyday life.
Stage 6 Biotechnology
Option 9.5.3
Perform a first-hand investigation to extract and identify
DNA from a suitable source.
Stage 6 Genetics Option
9.7.2
Outline the use of highly variable genes for DNA
fingerprinting of forensic samples, for paternity testing and
for determining the pedigree of animals.
Why Electrophoresis?
The movement of charged
particles in a fluid or gel
under the influence of an
electric field.
• Allows us to separate
complex mixtures.
• Molecules will migrate
towards the positive or
negative pole according
to their charge.
-
+
-
+
-
Electrophoresis Techniques
1. Using a Micropipettor
2. Preparing the experiment
– Preparing buffers
– Making an agarose gel
– Preparing DNA
3. Performing electrophoresis
– Preparing the tank and running the gel
– Loading a gel with dyes
– Loading a gel with DNA
4. Visualising the gel and analysing the data
Using a Micropipettor
What is a Micropipettor?
A tool for accurately
measuring liquid.
Why use a Micropipettor?
In Biotechnology usually
VERY small volumes are
required. This is the only
method for accurately
delivering small volumes.
Using a Micropipettor
Using a Micropipettor
Drawing up Liquid
A. Depress the plunger to position 2.
B. Place the tip in the solution.
C. Release the Plunger SLOWLY
A
B
C
Using a Micropipettor
Dispensing a Liquid
A. Gently touch the tip of to the bottom of the tube
B. Depress the plunger to position 3
C. Remove the tip from the tube BEFORE releasing the plunger.
B
C
Using a Micropipette
The volume can be
changed using the dial
on the top.
ALWAYS change tips
between every sample.
This prevents cross
contamination.
Using a Micropipette
TIME TO PRACTICE
Resources:
- 1 x 200 uL Micropipette
- 1 vial of dye
- Tips
- 1 piece of Parafilm
AIM: Pipette 20 uL of dye on to the parafilm. Try to make each
dot as even in size as possible.
Preparing the Experiment
To be prepared before the class:
1. Electrophoresis buffer
2. Agarose Gel
3. DNA solutions
Preparing Buffers
Electrophoresis buffer is supplied as 50 x TAE.
Prepare buffer by dilution to the correct
concentration with deionised water.
Agarose Gel
Agarose Gel
1. Weigh Agarose.
2. Add 1 x TAE.
3. Boil the soltuion in a microwave, stopping to
mix constantly. Continue to warm and mix
this solution until all solids are solubilised.
4. Allow to cool to 60⁰C. Add UV Stain.
5. Gently pour into a sealed tray
6. Place comb in the correct position.
Agarose Gel
TIME TO PRACTICE
Resources:
- 1 x 50 mL of Prepared agarose solution
- 1 x gel casting tray
- Pair of gel casting caps
- 1 x comb
Prepare the gel according to the instructions in the
manual.
Preparing DNA
DNA is supplied in individual tubes for each
student.
Thaw tubes as required and store at 4⁰C for up
to 24 hours.
Loading a gel with Dyes
TIME TO PRACTICE
Resources:
- 1 x prepared agarose gel
- 1 x electrophoresis tank
- Power Supply shared
- 1 x Micropipettor with tips
- Dyes
Load and run the gel according to the Manual.
Why Electrophoresis?
The movement of charged
particles in a fluid or gel
under the influence of an
electric field.
• Allows us to separate
complex mixtures.
• Molecules will migrate
towards the positive or
negative pole according
to their charge.
-
+
-
+
-
Why DNA Electrophoresis?
• DNA is negatively
charged. It will always
migrate towards the
anode.
• Agarose is a matrix that
will impede the migration
of the DNA.
• DNA will migrate
according to the size.
– Larger fragments of DNA
migrate more slowly than
smaller fragments of DNA
Why DNA Electrophoresis?
• This is commonly used technique is research
and analytical laboratories
• Most common method of comparing DNA.
This can be used in:
– Forensics
– Determining Genetic modifications in food
– Identifying organisms, such as pathogens
– Paternity
Loading a gel with DNA
TIME TO PRACTICE
Resources:
- 1 x agarose gel that you have prepared
- Electrophoresis tank shared
- Power supply shared
- 1 x Micropipettor with tips
- DNA
Load and run the gel according to the Manual.
Loading a Gel
1. Draw 20 uL of sample
using a pipette
2. Position the tip of the
pipette above the
centre of the well.
3. Depress the plunger
SLOWLY to position
4. Lift the tip from the
well before releasing
the plunger.
Preparing an Electrophoresis tank
1. Take the caps off the gel tray.
2. Place the gel tray in the tank.
3. Fill the tank with 1 x TAE so that the buffer is
just covering the surface of the gel.
NOTE: when using a Hexagel tank, students
should load gels first, then gels are placed in the
tank and buffer is gently poured on top until the
gels are covered.
Starting a Run
1. Put the lid on the tank. Red to Red/Black to
Black
2. Connect the tank to the power supply. Red to
Red/Black to Black
3. Set the Voltage to 150V.
4. Hit RUN!
Good separation should be achieved in about 30
mins.
Visualising gel
1.
2.
3.
4.
Place gel on UV transilluminator
Close safety shield
Switch on lamps
Take photo of gel
Staining Gel
1. Take 1 Insta-Stain card and place on gel.
2. Lightly wet card.
3. Allow gel to stain for at least 10 mins.
Plotting Results
Plotting results
Electrophoresis Techniques
1. Using a Micropipettor
2. Preparing the experiment
– Preparing buffers
– Making an agarose gel
– Preparing DNA
3. Performing electrophoresis
– Preparing the tank and running the gel
– Loading a gel with dyes
– Loading a gel with DNA
4. Visualising the gel and analysing the data
THANK YOU