Science Outreach
Gel Electrophoresis
This is a virus.
It is a λ bacteriophage or lambda phage
Viruses are simple
they consist of a
protein coat and…
they consist of a
protein coat and…
…..a circular piece of DNA
called a plasmid.
They need a host cell to reproduce.
The lambda phage
virus uses E Coli
bacteria as its host.
Virus inserts
it’s DNA into
the host cell.
Bacteria
reproduces
the viral
DNA.
The host cell becomes
a “virus factory”
E Coli bacterium and
many lambda phage
viruses…
Gel Electrophoresis:
Gel Electrophoresis:
separates DNA
fragments according to
their size
Gel Electrophoresis:
separates DNA
fragments according to
their size
gel
polysaccharide
strands form
microscopic gaps
DNA
fragments are
negatively
charged
DNA
fragments are
negatively
charged
Phosphate
backbone
DNA fragments migrate towards the
positive electrode.
DNA fragments migrate towards the
positive electrode.
SMALLER fragments migrate FURTHER
Plasmid in its
natural form:
Supercoiled
Plasmid in its
natural form:
Supercoiled
During
purification it
can be:
Nicked
Supercoiled
and sometimes
broken to form
DNA that is:
Linear
Nicked
Supercoiled
Linear
fast
Nicked
slow
movement through a gel
Restriction enzymes cut at
specific DNA sequences
DNA plasmid
There are many different
restriction enzymes.
There are many different
restriction enzymes.
You will only be using two:
EcoRI and HindIII
EcoRI
----------------GAATTC-----------------------------------CTTAAG-------------------
EcoRI
----------------GAATTC-----------------------------------CTTAAG-------------------
EcoRI
--------G
--------CTTAA
AATTC-----------G-----------
EcoRI
--------G
--------CTTAA
AATTC-----------G-----------
sticky ends
HindIII
----------------AAGCTT-----------------------------------TTCGAA-------------------
HindIII
--------A
--------TCGA
AGCTT-----------A-----------
HindIII
GCATAGTCTCAAGCTTTTGAAGA
CGTATCAGAGTTCGAAAACTTCT
HindIII
AGTCTCA
AGCTTTTGAAG
TCAGAGTTCGA
AAACTTC
Bacteriophage
plasmid
Working with small volumes
• Need to be accurate
• 1000 microlitres (µL) = 1 millilitre (mL)
micro
pipette
Eppendorf
tube
Working with small volumes
You may have to shake
the Eppendorf tube to get
all the liquid to the bottom
20
200
Micropipettes
measure small volumes of liquid in
microlitres ( µL )
1000 µL = 1 mL
tip ejector button
volume adjustment
knob
plunger
tip
volume
indicator
10 µL
Volume indicator
20.0 µL
3 positions for the plunger
Rest
position
First stop
Second stop
Get liquid INTO the pipette
Get liquid INTO the pipette
use a new tip for each sample
1. Rest
position
(attach tip)
2. First stop
1. Rest
position
(attach tip)
2. First stop
1. Rest
position
(attach tip)
3. Immerse tip in the liquid
and release plunger
(back to rest position).
2. First stop
1. Rest
position
(attach tip)
3. Immerse tip in the liquid
and release plunger
(back to rest position).
Wait a second for liquid
to be sucked into tip.
Get liquid OUT of the pipette
press plunger to the first stop
press plunger to the first stop ..then to the second stop
Remove tip
using the tip
ejector button.
Remove tip
using the tip
ejector button.
Use a new tip
for the next
sample…..
Practise filling and emptying
the pipette with 5, 10 and 20 µL
of water.
The enzyme digestion has been done for you.
Tube “L” has 15 µL of DNA in it.
All other tubes have 10 µL .
X = Unknown
sample
X
U = uncut
plasmid
E = EcoRI +
plasmid
U
E
H
D
L
L = DNA Ladder
(DNA fragments
of known sizes)
H = HindIII +
plasmid D = “double”
EcoRI + HindIII
+ plasmid
Add 5 µL of water
to the DNA
Ladder.
Add and 10 µL of
water to all other
DNA samples.
This will make them all up to 20 µL
Place all the eppendorf tubes in hot water (65 °C)
for 5 minutes
…then in ice for 3 minutes.
Run the pre-run program (2 minutes)
Remove the comb from the gel
Remove the comb from the gel
Add 20µL of each DNA
sample into a well of the
gel
Carefully rest the
pipette tip on the
inside edge of the
well
Fill the empty
wells with 20 µL
of water
Run the gel for 15 or 30 minutes
Place on the viewer
What to
expect….
Size
(base pairs)
DNA Ladder
Standard, known
DNA lengths.
HindIII
EcoRI
Both
The
unknown
sample
“X” will
be one of
these.
HindIII
EcoRI
Both
Lane 2:
Uncut Lambda DNA
Lane 3:
Lambda DNA cut with
EcoRI
Lane 4:
Lambda DNA cut with
HindIII
Lane 5:
Lambda DNA cut with
EcoRI and HindIII
Lane 6:
DNA Ladder
What can we use this technique for?
A
N
g
D
n
i
t
n
i
r
p
r
e
Forensics
g
n
i
f
Pate
rnity
case
s
Diagno
se gen
etic
diseas
es
n
o
i
t
a
l
o
s
i
d
n
a
n
o
i
t
a
d
r
n
a
a
p
e
s
S
g
u
r
d
r
o
f
A
N
g
D
n
f
i
r
o
e
e
n
i
g
d
n
e
n
c
i
a
t
n
gene
o
s
i
t
e
a
m
v
r
m
e
a
s
r
n
g
o
r
Co
p
g
n
i
d
Evolution
e
ary
bre
Res
biology
earc
h
Science at University
Year 9-11
Science
Year 12-13
Biology
Chemistry
Physics
Maths
University
Bachelor of Science – 3 years
Honours / Masters– 1-2 years
Ph.D. Degree - ~4 years
Teacher training
Polytechnics
Other...
Postgraduate
Diploma
(teaching, other…)
Employment
Science at University
Conservation
Astronomy
Biochemistry
Geography
Engineering
Biology
Forestry
Antarctic Studies
Psychology
Chemistry
Physics
Geology
Soil Science
Electronics