Laboratory Manual
PROJECT 9:
Investigating the Role of Caveolae in
Aggressive Prostate Cancer
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
1
Acknowledgements
SPARQed is a collaboration between The University of Queensland Diamantina Institute and The
Queensland Government’s Department of Education and Training. It exists due to the hard work of the
SPARQ-ed Regional Reference Group (Kerry Holst, Regan Neumann, Associate Professor Brian Gabrielli,
Associate Professor Nigel McMillan, Dr Peter Darben, Doreen Awabdy, Cheryl Capra, Andrew Rhule,
Michael Sparks, Patrick Trussler and Jacki Wilton).
The Caveolin project was developed by Dr Michelle Hill.
The immersion research program was adapted for student use by Dr Peter Darben, under the supervision
of Dr Michelle Hill.
Risk assessments were developed with the assistance of Paul Kristensen, Maria Somodevilla-Torres and
Jane Easson.
Cover Image :
Logo design by Danielle Fischer.
Many thanks to all in the Hill Lab whose patience and support made this happen.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
2
Table of Contents
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prostate Cancer Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cholesterol and Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Caveolins and Caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Signal Transduction Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Project Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfection of Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How to Use This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Culture Media and Other Solutions . . . . . . . . . . . . . . . . .
Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Lipoplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfection of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Serum Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microscopy and Interpretation of Results . . . . . . . . . . . . . . . . . . . . . . .
Recovery of DNA Plasmids Using an Akaline Lysis Mini-Plasmid Preparation
QIAprep Spin DNA Purification System (QIAGEN) . . . . . . . . . . . . . . . .
Restriction Digests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
4
4
4
5
5
7
10
10
10
11
14
15
16
16
17
18
19
20
21
21
22
24
Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A – Some Basic Knowledge . . . . . . . . . . . . . . . . . . . . . . . . .
A1 – DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A2 – DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A3 – Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A4 – Transcription & Translation . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix B – Cell and Molecular Biology Techniques . . . . . . . . . . . . . .
B1 – DNA Restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B2 - Agarose Gel Electrophoresis for DNA . . . . . . . . . . . . . . . . . . . .
B3 – Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix C – Standard Operating Procedures . . . . . . . . . . . . . . . . . .
C1 – Using a Micropipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C2 – Using a Bench Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C3 – Using a Haemocytometer . . . . . . . . . . . . . . . . . . . . . . . . . . .
C4 – Using a Biological Safety Cabinet . . . . . . . . . . . . . . . . . . . . . . .
C5 – Using the Fluorescence Microscope . . . . . . . . . . . . . . . . . . . . .
Appendix D – Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
27
27
29
31
36
39
39
40
41
46
46
51
53
56
57
63
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
3
Background Information
Prostate Cancer
Prostate cancer is the second most common cause of death in men in western countries. Originating in
glandular tissue within the prostate, most prostate cancers are slow growing and often patients will die
of other causes ‘with’ prostate cancer rather than ‘from’ the disease.
The causes of prostatic cancers vary widely, however age and genetic factors play an important role.
Prostate cancer may arise from normal benign changes to the prostate which occur with age, and
autopsies on men who have died from other causes have found that a high proportion was harbouring
prostatic cancers without showing any symptoms. However on occasion, prostate cancers may grow
aggressively and spread to other parts of the body (metastasize). Such cancers are difficult to treat and
many prove fatal when the cancerous cells form tumours which disrupt the function of other organs.
Prostate cancers can be treated by surgical removal of the tumour, although this can be accompanied by
loss of normal function of the uro-genital tract. Since many prostate cancers are stimulated by the levels
of certain hormones, targeting these hormones or their action on the cancerous cells can help slow the
development of the disease.
One group of hormones which can have a significant effect on the development and progression of
prostate cancers are the androgens, steroid hormones involved in the development and maintenance of
male characteristics and function. The best known androgen is testosterone, and most androgens are
derivatives of this compound. Androgens are known to stimulate the proliferation of prostate tissue
cells, and if the response to androgens is abnormal, this proliferation may lead to hyperproliferation and
cancer.
Therapies which target the action of androgens are called androgen ablative therapies. These may
involve the use of drugs with a similar structure to the androgens (analogs) but which do not have the
stimulatory effect, removal of androgen producing organs like the testes (castration), or using drugs
which lower the levels of androgens in the body (chemical castration). Most prostate cancers respond
well to these therapies, however a significant subset of cancers return after treatment, and thereafter
are completely unaffected by further androgen ablative therapies (the castrate resistant phenotype).
These cancers are difficult to treat and may progress to fatal conditions.
Prostate Cancer Metastasis
Localised prostate cancer is highly curable, usually through removal of the tumour and the use of
androgen ablative therapies. Serious problems arise when these cancers metastasize. Metastasis is
caused by specific changes within the cancerous cells which alter their normal function and behavior.
Normal cells tend to remain in one place, a trait assisted by the cells joining together through adhesion
structures found on their exterior surfaces. Cancerous cells lose this property, allowing them to break
away from the cell mass. In addition, cancerous cells produce proteases to dissolve the non-cellular
matrix in tissue, allowing them to move more freely. Once a cancer cell is free from the tissue, it can
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
4
travel to other parts of the body through the bloodstream. Normal cells do not grow unless they are
anchored to other cells or to a matrix in the tissue. Cancer cells have the ability to grow without these
attachments.
Cholesterol and Prostate Cancer
High blood cholesterol has been linked to increased risk of advanced prostate cancer. There are two
possible mechanisms for this. Cholesterol is a precursor of androgens like testosterone, and it has been
hypothesized that prostate cancer cells can produce androgens which act in an autocrine manner to
stimulate their own proliferation in castration-resistant prostate cancer. Secondly, cholesterol is an
essential component of cellular membranes and it is particularly enriched in special membrane domains
termed lipid rafts. Lipid rafts are regions of the cell membrane with a high level of sphingolipids,
cholesterol and phosphatidylserine. These regions are known to regulate membrane traffic as well as
communication of signals from outside of the cell (signal transduction). Changes in the function of lipid
rafts have been reported in prostate cancer cells, and may correlate to increased metastasis.
Caveolins and Caveolae
The caveolins are a family of proteins which are associated with structures in the cell membrane called
caveolae (latin for “little caves”). Caveolae are specialised forms of lipid rafts. The caveolin proteins
embed in the phospholipid bilayer of the membrane and distort it so that it forms small flask-like pits.
These pits (the caveolae) are often rich in protein receptors involved in signal transduction pathways and
serve as areas in which the ligands to which the receptors bind can concentrate, thus regulating the
signal transmitted by the pathway.
a)
b)
c)
Cavin 1
Figure 1 – Structure of Caveolin-1 showing (a) single molecule, (b) dimer with Cavin-1 and (c) oligomer
Caveolae are produced at the Golgi apparatus when caveolin joins together in small groups (oligomers)
and binds to cholesterol. These vesicles move to the cell surface and merge with the cell membrane. An
associated protein called Cavin-1 binds to caveolin-1/cholesterol, forming the caveolar shape and
allowing normal caveolin-1 function (Figure 2).
The relationship between caveolins and cancer is complex, with expression of the proteins associated
with tumour suppression in some cancers, and tumour promotion in others. In prostate cancer, the level
of caveolin-1 correlates strongly with tumour stage and grade. Research has found that prostate tumours
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
5
with higher levels of caveolin-1 were more likely to be aggressive tumours which recurred after
treatment and which were resistant to androgen ablative therapies. Conversely, in experimental
animals where the expression of caveolin-1 has been knocked out, castrate resistant cancers have been
shown to regress to being androgen sensitive again. In addition, when Cavin-1 is absent, caveolae do not
form and this state is also associated with aggressive cancers. In this study, we are investigating the
possibility that abnormal caveolin-1 function in advanced prostate cancers is caused by missing cavin-1,
and high cholesterol further exacerbates this abnormal caveolin-1 function in the spread of prostate
tumours (metastasis).
a)
b)
c)
Figure 2 – Interaction of Caveolin-1 with the
cell membrane. In the absence of Cavin-1
(a), caveolin-1 oligomers embed in the cell
membrane but do not form caveolae. The
presence of Cavin-1 allows caveolin-1 to
interact with the cell membrane to form
caveolae (b). The association of the
caveolins oligomers, the lipid bilayer,
sphingolipids and cholesterol is shown in (c).
(after Williams and Lisanti (2004) The
Caveolin Proteins. Genome Biology 5, 214)
Phospholipid
Sphingolipid
Cholesterol
Because it is associated with the cell membrane and with signal transduction, Caveolin-1 is involved with
both the migration of cells away from tissue and anchorage independent growth. As a result, not only
does Caveolin-1 expression and function correlate with androgen independent cancers, it is also a strong
indicator of the likelihood that the cancer will metastasize.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
6
Signal Transduction Pathways
For a cell to respond to an outside stimulus, that stimulus must somehow find its way across the
membrane and into the part of the cell which mediates the response (eg. the nucleus). The cell
membrane provides an effective barrier to the entry (or departure) of most materials, so cells use
methods to transmit a signal across this membrane, often without the stimulating material actually
crossing the membrane itself.
One method for transmitting this stimulus is through signal transduction. This involves transmembrane
proteins called receptors. A typical cell receptor has extracellular domains, membrane domains (often
rich in hydrophobic amino acids) and intracellular domains. The extracellular domain has a tertiary
structure which enables it to bind to a specific compound (called a ligand) which acts as the stimulus for
the signal. When the extracellular domain binds to the ligand, it causes a conformational change in the
protein which allows the intracellular domain to carry out a function inside the cell. For example, if the
receptor is a kinase, this involves the attachment of a phosphate group to another protein. The
phosphorylation of this protein may allow it to change another protein and so on, until the signal
generated by the binding of the ligand outside the cell is passed on to the parts of the cell which
generate the response. This sequence of events, each mediated by a change to a protein, is called a
signal transduction pathway or cascade.
An example of a signal transduction pathway is
Why All These Abbreviations ?
the Ras-MAPK pathway 1 (Figure 3). In this
pathway, a receptor binds to an extracellular
Cell signaling cascades are incredibly complex and
ligand (eg. the hormone epidermal growth
may contain hundreds of intersecting pathways
factor, or EGF). This causes the receptors to join
and interacting proteins. Protein names are
together to form a dimer and to attach
abbreviated to make them easier to handle, but
phosphate
groups
to
itself
the number of abbreviations can become
(autophosphorylation). Through an adaptor
overwhelming. To add to the confusion, names
protein, this complex activates Ras, which binds
change – MAPK originally stood for “Microtubule
to and phosphorylates another protein Raf. The
Associated Protein Kinase”, but now stands for
Ras/Raf complex then phosphorylates MEK and
“Mitogen Activated Protein Kinase”. Should you
the phosphorylated MEK phosphorylates MAPK.
ever become a cell biologist, you will need to know
Activated MAPK then activates a series of other
what each of these are and what the abbreviations
proteins which encourage the expression of
stand for, however for the purposes of this project,
genes which promote cell proliferation. Under
it is sufficient just to know them by their
normal circumstances, this pathway promotes
abbreviations and the role they play in the cell.
the proliferation of cells when they are needed
(eg. for growth or to repair cellular damage).
However, abnormalities in this pathway can lead to uncontrolled cell proliferation, which is one of the
hallmarks of cancer. As a result, many cancer researchers study this pathway to try to find elements in it
which may lead to new therapies.
1
The first MAPKs identified, MAPK1 and MAPK2, were originally called Extracellular Signal-regulated Kinases
(ERK), and this term is sometimes still used to describe these enzymes and the pathways which contain them.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
7
EGF
Ras
GDP
EGF
Receptors
P
P
P
P
P
P
Ras
Ras
GTP
GTP
Raf
P
Adaptor Proteins
Raf
Autophosphorylation
MAPK
P
MEK
P
MAPK
P
P
Transcription factors which
promote the expression of
proteins which encourage
cell
growth
and
proliferation
Nucleus
Figure 3 – Simplified Diagram of the MAPK Pathway (after Kleinsmith – Principles of Cancer Biology)
One effect of ERK signaling with particular significance to metastasis is to regulate the removal of
adhesion points between the cell membrane and the extracellular matrix. Increases in ERK signaling have
been shown to promote cell migration by limiting these focal adhesions and so is correlated to
metastasis.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
8
MEK
Research has identified members of a protein complex associated with ERK signaling in lipid rafts
obtained from prostate cancer cell lines which show abnormalities in the formation of caveolae. These
proteins – MEK Partner 1 (MP1), MAPK Scaffold Protein I (MAPKSP1 or p14) and Pdro regulate MAPK
signaling by separating or bringing components of the pathway together. In doing so, they help to
regulate cell migration. Pdro also appears to attach to the protein actin, a component of the
cytoskeleton which plays a vital role in cell mobility. The exact localization of these proteins has yet to be
elucidated, as the lipid raft preparations were derived from whole cells. MP1 has been reported to
associate with intracellular membrane bound bodies called endosomes. What is not known is whether
there is a difference in localization when caveolae formation is affected, and when lipid raft is altered by
changes in cholesterol levels. Since the lack of caveolae in some cancer cell lines affects their signaling
pathways, we need to know whether this is because there is a change in the localization of the
components of these pathways. If these signaling pathways influence the ability of the cells to migrate
and metastasize, an understanding of these mechanisms may help us to develop treatments which
overcome or reverse abnormalities found in cancer cells and so help to prevent the more serious
outcomes of prostate cancer.
References :
Bennett, N. et al (2009) Androgen Receptor and Caveolin-1 in Prostate Cancer. IUBMB Life 61(10), 961970. [PMID: 19787702]
Hill, M. et al (2008) PTRF-Cavin, a Conserved Cytoplasmic Protein Required for Caveola Formation and
Function. Cell 132, 113-124. [PMID: 18191225; PMCID: PMC2265257]
Hoshino, D. et al (2009) A Novel Protein Associated with Membrane-type 1 Matrix Metalloproteinase
Binds p27kip1 and Regulates RhoA Activation, Actin Remodeling, and Matrigel Invasion. The Journal of
Biological Chemistry 284(40), 27315–27326. [PMID: 19654316]
Kleinschmidt, L. (2006) Principles of Cancer Biology. Benjamin Cummings, San Francisco.
Williams, T. and Lisanti, M. (2004) The Caveolin Proteins. Genome Biology 5, 214. [PMID: 15003112]
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
9
Experimental Procedures
Project Overview
In this project, you will be using one of two lines of prostate cancer cells which do not produce normal
caveolae. After transfecting these cells with the genes for Cavin-1 and MP1, you will be observing the
formation of caveolae in the absence of cholesterol, with cholesterol present at normal levels and in the
presence of high levels of cholesterol. Using immunofluorescence targeting the MP1, you will examine
the localization of elements of the ERK pathways in cells which produce caveolae and in cells where
caveolae have not formed properly.
Transfection of Cancer Cells
Transfection is the introduction of genetic material into eukaryotic cells. It is usually done through
liposomes – small “bubbles” of lipid bilayer which mimic and merge with the cell membrane. This form of
genetic modification is widely used in cell biology, particularly when observing the effects of single
genes. For example, in cells where a particular gene has been knocked out or is abnormal, normal
function can be returned by the transfection of that gene.
You will be using two prostate cancer cell lines. Both are deficient in normal caveolae formation. PC3
cells are an aggressive, androgen-independent cell line that express Caveolin-1, but cannot produce
caveolae because they do not express Cavin-1. 22Rv1 cells are androgen-dependent and do not express
either Caveolin-1 or Cavin-1. If the gene for Cavin-1 is introduced into PC3 Cells, Cavin-1 is expressed by
the cells and caveolae can be observed in them. However, the lack of Caveolin-1 in the 22Rv1 cells
means that even if the Cavin-1 gene is introduced, they still will not form caveolae.
Caveolae are difficult to visualize without an electron microscope. However if the proteins associated
with the caveolae (such as Cavin-1) can be tagged with a fluorescent label, the presence of the caveolae
can be visualized using fluorescence microscopy. In this project, the gene for Cavin-1 which will be
transfected into the test cells has been joined onto a gene
a)
b)
for the jellyfish derived green fluorescent protein (GFP).
This produces a version of Cavin-1 which fluoresces green
under ultraviolet light.
In cells which do not produce caveolae, Cavin-1 is
distributed evenly throughout the cell (Figure 4a).
However, when Cavin-1 binds to Caveolin-1 and caveolae
are formed, the fluorescence takes on a spotty or
“punctuate” appearance (Figure 4b).
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
Figure 4 - Distribution of Cavin-1-GFP in
cells lacking caveolae (a) and with
caveolae (b) (Images courtesy M. Hill)
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
10
Immunofluorescence
In addition to observing whether caveolae are formed, we are also interested in observing the
distribution of components of the ERK signaling pathways in the presence and absence of caveolae. The
marker of these proteins we will be looking for is MP1. Under normal circumstances, we could use a
labelled antibody directed against this protein, however such an antibody has yet to be produced.
Instead, we have added a short tag (a fragment of the myc gene) to the MP1 gene and will be using an
antibody targeted to it. The label attached to this antibody will be blue rather than green, allowing us to
observe any colocalisation of Cavin-1 and MP1.
Since MP1 is localized to endolysosomes, we will also use a “lysosomal tracker” a material labeled with a
third colour (red) which is taken up through endocytosis to indicate the location of endosomes in the
cytoplasm.
A summary of the procedure for the week is provided in Figure 5.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
11
PC3 or 22Rv1 Cells Provided
Plated onto Coverslips.
Lipofectamine
Plasmid
Transfect Cells
with Lipoplexes
Lipoplexes
Incubate @ 37°C for 4 hrs
Change media and
incubate O/N @ 37°C
Cholesterol Depleted Serum
Normal Serum
Cholesterol Loaded Serum
Treat with
anti-myc
primary and
labeled secondary
antibody for
Immunofluorescence
Fix in PFA
Observe using
fluorescence
and confocal
microscopy
Figure 5 – Caveolae Project Protocol
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
12
TUESDAY
WEDNESDAY
30 min incubation with
lysotracker
Fix in PFA
Treatment with primary and
secondary antibody for
Immunofluorescence
Restriction digest
THURSDAY
Library Research
Skills Workshop
Confocal microscopy
FRIDAY
Image processing and
preparation of
presentation
13
MONDAY
Treat cells with cholesterol
depleted, normal and
cholesterol loaded serum
(overnight)
Mini-Prep to recover plasmid
DNA (Alternate Program)
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
Combine oligofectamine
and plasmid to create
lipoplexes (20 min at RT)
then add to cells
Incubate at 37°C
for 4 hours
Change media and
Incubate O/N at 37°C
Bioinformatics Workshop
Agarose gel electrophoresis
Figure 6 - Week Overview
How to Use this Manual
Throughout this section you will see a series of icons which represent what you should do at each point.
These icons are:

Write down a result or perform a calculation.
Prepare a reaction tube.

Incubate your samples.
Some solutions need to be made up fresh. The methods for doing this can be found in
purple boxes to the right side of the text.
When you are asked to deliver a set volume, the text will be given a colour representing the colour of the
micropipette used:
e.g.
750µL
Use the blue P1000 micropipette (200-1000µL)
100µL
Use the strong yellow P200 micropipette (20-200µL)
15µL
Use the pale yellow P20 micropipette (2-20µL)
2µL
Use the orange P2 micropipette (0.1-2µL)
Sometimes the exact volumes of solutions needed will not be known until the results of a previous stage
of the experiment are completed. Therefore, some of our protocols do not have exact volumes listed.
During the course of the experiment, and with the aid of your tutors, you will be asked to calculate the
volumes needed using dilution factors. In early examples, the manual will have step by step procedures
taking you through the process. However by the end of the week you should be confident to do the
calculations on your own.
In this project, you will be working in one of three groups of 4 or 5 students. Each group will be using
either the Cavin-1 deficient PC3 prostate cancer cells PC3 or the Cavin-1/Caveolin-1 deficient 22Rv1
prostate cancer cells. Make sure you record which group you are in below :
Cell Line : __________________________________
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
14
Preparation of Culture Media and Other Solutions
Unlike bacteria, mammalian cells require a culture medium featuring a range of complex growth factors
and nutrient sources. The exact make-up of the culture medium depends on the requirements of the
cells to be cultured. Cell culture media are usually made up fresh, by combining a commercially sourced
basal medium with supplementary nutrients, growth factors and antibiotics to prevent overgrowth by
bacteria.
Commonly used ingredients in cell culture media include :
Basal Medium – a commercial broth containing salts, sugars, buffers and other nutrients needed
by all cell culture lines. These media are often coloured pink due to the presence of a pH
indicator. As the cells metabolise, they produced wastes which change the colour of the medium
to yellow, which can be used as an indicator of when the medium should be changed. The basal
medium used in this project is DMEM (Dulbeco’s Modified Eagle’s medium).
Foetal Bovine Serum (FBS, also called Foetal Calf Serum) – the liquid component of clotted blood
from a bovine foetus. This provides a complex series of nutrients required by the cultured cells.
Pen/Strep/Glut – a mixture of the antibiotics penicillin and streptomycin, and the amino acid
glutamine. The antibiotics prevent the growth of bacteria which may contaminate the medium
and interfere with the growth of the cells. The glutamine supports the growth of rapidly growing
cells.
Sodium Pyruvate – pyruvate is the product of glycolysis and is the feedstock for the citric acid
cycle in cellular respiration. It is included as an additional energy source.
HEPES Buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) – a buffer which is particularly
efficient in resisting the changes in pH caused by the release of carbon dioxide by cells
Each pair will prepare their own culture medium to use through the course of the week.
Cell Culture Medium
To a 500ml bottle of DMEM, add the following components :
Culture
Medium
FBS
Pen / Strep /
Glutamine
50mL
5mL
Sodium
Pyruvate
5mL
Place the bottle of medium in the 37°C waterbath to bring it to the correct temperature.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
15
Phosphate Buffered Saline (PBS)
You are provided with PBS at which is ten times (10X) more concentrated than what is needed – this is
known as a stock solution. Stock solutions are made at a higher concentration and then diluted as
necessary to provide working solutions (1X). The use of stock solutions saves us the bother of having to
make up new solutions every time they are needed.
You need to make your working solution of PBS by diluting it appropriately in water. You are provided
with a 500mL sterile “Baxter” water bottle for this purpose. The process for working out what volume to
add to the Baxter water is as follows :
Total volume
= 500mL
1/10 of 500mL = 500 ÷ 10 = ____ mL
volume of 10X stock needed is ____ mL
Remove this volume from the Baxter water bottle
Add this volume of 10X PBS stock to the Baxter water bottle
Transfection
The first stage in the project involves transfecting your cells with plasmids containing the genes for GFP
tagged Cavin-1 and myc tagged MP1. You will be using freshly prepared lipoplexes for this purpose.
Preparation of Lipoplexes
You are provided with a solution of DNA with the concentration stated on the vial. In order to have
enough DNA for our experiments, you will need to have 2μg of DNA total in a total volume of 100μL.
To calculate how to dilute your DNA to the correct concentration :
Initial concentration of DNA = _____ μg / μL
Number of μL containing 2μg DNA = _______
Volume of Opti-MEM diluent required = 100μL – Volume calculated above
= ______ μL
Dilute the DNA solution in Opti-MEM using the volumes calculated above

Incubate the solution for 5 minutes at room temperature
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
16
You will need twice the volume of Lipofectamine (LF2000) as you have μg of DNA in the diluted
sample
μg DNA in diluted sample = _______ μg
μL LF2000 required (above value x 2) = _______ μL
Volume Opti-MEM diluents required
= 100μL - Volume calculated above
= _____ μL
Dilute the LF2000 in Opti-MEM using the volumes calculated above

Incubate the solution for five minutes at room temperature
Combine the diluted DNA solution and the diluted Opti-MEM solution and vortex

Incubate the combined solutions for 20 minutes at room temperature
Transfection of Cells
You have been provided with a 6 well culture plate containing the cell line you will be working on. These
plates were seeded with cells yesterday and also contain a small glass coverslip at the bottom of the
well. After seeding, as the cells fell to the bottom of the well, some fell onto the coverslips, attached and
started to grow and multiply. This allows us to easily remove the cells from the wells when the time
comes to mount them on the slide simply be removing the coverslip.
Cells grown in culture generally multiply until they touch neighbouring cells. This means that after a
certain amount of time, the cells form a mono-layer on the bottom of the well or coverslip. Cells that
have done this are said to be confluent. Allowing cells to grow until they reach a sufficient degree of
confluence ensures an adequate coverage of the coverslip without negative effects on the cells caused
by overcrowding. We have incubated the cells so that they now cover 50% of the coverslip (50%
confluency).
Without removing the lid from your culture plate, observe the cells under a plate microscope to
ensure that they are sufficiently confluent.
In the tissue culture hood :
Using the vacuum line, remove the old culture medium from each of the wells
Add 2mL of Opti-MEM reduced serum medium to each of the wells
Add 30μL of the lipoplex suspension to each of the wells

Incubate the plate in the 37°C CO2 incubator for 4 hours
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
17
Remove the Opti-MEM from each of the wells using the vacuum line
Add 2mL of cell culture medium to each of the wells

Incubate the plate in the 37°C CO2 incubator overnight
Serum Treatment
Cholesterol plays an essential role in the formation of caveolae. To this end, in this project, we will be
seeing the effect of depleting or raising the level of cholesterol available to cells for caveolae formation.
In the body, cells recruit cholesterol from the blood. When grown in culture, cholesterol is obtained
through the serum that forms part of the culture medium. If grown in the presence of serum which has
low cholesterol, the cells lose cholesterol from their membranes to the serum. Conversely, when grown
in the presence of serum containing high levels of cholesterol, additional cholesterol is incorporated into
the cell membrane. In this project, we will be growing our transfected cells in the presence of serum
from which cholesterol has been depleted (low cholesterol serum), normal serum and serum to which
extra cholesterol has been added.
It is important to know which wells contain which treatment. In the diagram below, indicate the
treatment added to each well (you will be doing duplicate treatments) :
Place your 1X PBS on ice
Remove the culture medium from each of the wells using the aspiration line
Add 2mL of the appropriate serum to each of the wells

Incubate the plates overnight in the 37°C CO2 incubator
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
18
Prepare the Lysotracker by adding 1μL to each 1000μL of Opti-MEM – you will need to prepare
enough Lysotracker / Opti-MEM for 6 wells (ie. 6 x 2mL = at least 12mL)
Remove the serum from each well using the aspiration line
Add 2mL of Lysotracker / Opti-MEM to each of the wells

Incubate the plate in the 37°C CO2 incubator for 30 minutes
When the time is up, place the cells on ice and remove the liquid from each well using the
aspiration line
Wash the cells by adding 2mL of ice cold 1X PBS to each well and removing with the aspiration
line
Fix the cells by adding 2mL of 2% PFA to each well

Incubate the plates at room temperature 20 minutes
Wash the cells three times in 1X PBS as described above
Retain the plates in 1X PBS overnight at 4°C
Immunofluorescence
Remove the liquid from each of the wells using the aspiration line
Add 1mL of block/permeablisation solution (1X PBS containing 1% BSA and 0.1% Triton X100) to
each of wells

Incubate the plate at room temperature for 30 minutes
Set up primary antibody spots (diluted 1 in 50 in 1% BSA) as demonstrated by your tutor and
place each coverslip face down on separate spots

Incubate the coverslips under aluminium foil for 1-2 hours at room temperature
Wash the coverslips three times in 1X PBS, 5 minutes each time
Set up secondary antibody spots as previously described and place each coverslip face down on
separate spots

Incubate the coverslips under aluminium foil for one hour at room temperature
Wash the coverslips three times in 1X PBS, 5 minutes each time
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
19
Wash the coverslips twice in water for 5 minutes each time
Mount the coverslips as instructed
Microscopy and Interpretation of Results
Your cells can be observed using either routine fluorescence microscopy or confocal microscopy. Normal
fluorescence microscopy observes cells as a whole. Materials localized to the membrane (like caveolae)
will be observed as appearing throughout the cytoplasm and even over the nucleus, as regions of the
cytoplasm may overlie it. Therefore, using this method it is difficult to determine whether the
fluorescence is confined to the cytoplasm or localized to the membrane. In confocal microscopy, a
narrow “slice” of the cell is imaged which is thinner than the cell. This means that materials found
throughout the cytoplasm will have a diffuse distribution everywhere except for the nucleus (which will
appear as a black hole), while materials localized along the membrane will appear as a coloured halo
around the cell.
You will be looking for three different cellular components, each labeled with a different colour :
Cavin-1 will be labeled green by the presence of the GFP tag
MP-1 will be labeled blue by immunofluorescence targeting its myc tag
Endosomes will be labeled red by the Lysotracker
Each of these components will be imaged separately and then combined to provide a three-colour
image.
When observing your specimens, you will need to look for
The presence of each of the labeled components and their location
The distribution of each of the components – an even diffuse pattern suggests no caveolar
localization, while a punctuate (“spotty”) distribution suggests localization to caveolae or
endosomes
Any co-localisation – are some components found in the same places as others. This is most
easily seen in the separate colour channel images, however it may also be seen by a change of
colour (eg. green and red colocalised appears yellow)
Any differences between transfected and untransfected cells – not all cells take up the lipoplexes
and these should be lacking in either the green or blue labels (or even both). Use these cells as
negative controls for the experiment (ie. what would happen if the procedure didn’t work).
Take images of indicative fields for each of your coverslips. If time permits you may even attempt to
quantitate each of the signals.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
20
Supplementary Activity – Recovery of DNA Plasmids Using an Akaline Lysis Mini-Plasmid
Preparation
The DNA plasmids used to transfect the cells in this project were recovered from culture of bacteria (E.
coli). Each plasmid had been constructed using cloning technology and then used to transform the E. coli.
Since bacteria make copies of any plasmids they contain when they multiply, this makes these cultures a
convenient “plasmid” factory for whenever we need large quantities of the plasmids for our research.
In order to obtain pure solutions of the plasmids for transfection, we need to recover them from the
bacterial cultures. In laboratories, this is done using either “midi-preps” (for large quantities of DNA) or
“mini-preps” (for smaller quantities of DNA). Both techniques use the same principles :
Growth of transformed bacterial cultures
Lysis (“bursting”) of the bacterial cells to release their DNA using an alkaline solution
Neutralisation of the alkaline solution and precipitation and removal of proteins using an acidic
solution
Using a commercial column to bind the plasmid DNA but allow non-plasmid DNA and other
cellular components to wash through
Elution and collection of the plasmid DNA
In this project, as a supplementary activity, you will be using a commercial alkaline lysis mini-plasmid
preparation kit to recover the plasmids you used to transform your cells from a culture of transformed E.
coli. This will provide more plasmids for the research group to use in future transfections.
NOTE: While this strain of E. coli has a low
risk associated with it, all bacterial culture
work should be done using strict aseptic
technique to prevent escape of the
organism or cross-contamination.
QIAprep Spin DNA Purification System (QIAGEN)
Production of Cleared Lysate
Transfer 1mL of each overnight culture into separate Eppendorf tubes and centrifuge at
8,000rpm for 5 minutes.
Remove supernatant from each of the tubes and resuspend in 250µL Buffer P1. Ensure that there
are no cell clumps visible after resuspension of the pellet.
Add 250µL Buffer P2 to each sample and invert 4-6 times to mix. The solution should turn a blue
colour – mix until this colour is uniform throughout the tube
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
21
Add 350µL Buffer N3 and invert 4-6 times to mix. The solution should lose its colour and become
cloudy as proteins precipitate. Mix until all of the blue has disappeared.
Centrifuge at 13,000rpm for 10 minutes at room temperature.
Binding of Plasmid DNA
Insert a QIAprep spin column into a centrifuge tube for each sample.
Transfer the supernatant from the lysis stage into the spin column.
Centrifuge at 13,000rpm for 1 minute at room temperature.
Discard the flowthrough in the centrifuge tube and reinsert the column into the centrifuge tube.
Washing
Add 500µL Buffer PB (binding buffer) to the Spin Column.
Centrifuge at 13,000rpm for 1 minute.
Discard the flow through in the centrifuge tube and reinsert the column into the centrifuge tube.
Repeat wash steps above with 750µL Buffer PE (wash solution).
Centrifuge at 13,000rpm for 2 minutes at room temperature.
Elution
Transfer the spin column to the sterile Eppendorf tube, taking care not to transfer any of the
wash solution.
Add 20µL Buffer EB (elution buffer) to the spin column.

Incubate at room temperature for 1 minute.
Centrifuge at 13,000rpm for 1 minute at room temperature and discard the column. Samples can
be stored at -20°C or below.
Restriction Digests
To ensure that we have successfully recovered the plasmid, we need to demonstrate its presence using
gel electrophoresis. Since the plasmid is a circular length of DNA, we must linearise it before it is run on a
gel. This involves cutting the plasmid once using a restriction digest.
Restriction endonucleases are enzymes which cut the DNA strand at a specific sequence of bases. By
careful selection of the enzymes, we can choose to cut a piece of DNA at a single site to give us a single
linear fragment.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
22
Restriction digests have a number of common components in addition to the DNA to be digested and the
restriction enzyme :
Buffer – maintains the correct pH and ion concentrations to ensure optimal conditions for
enzyme activity. Buffers are often specific for the enzyme used (there are four common buffers
used for most restriction enzymes – make sure you use the correct one for your enzyme). Buffers
are supplied at “10X”, meaning they are 10 times more concentrated than they need to be. The
volume of buffer added must be one tenth of the total volume.
BSA (Bovine Serum Albumin) – a protein product derived from the blood of cows, BSA serves to
stabilize the enzyme. Not all restriction endonucleases need BSA.
Water – to make up to a workable final volume.
Normally, when preparing the digest solution, the exact volumes used depend on the initial
concentration of the DNA you are working with. Since we do not know what this is, we will start using a
volume of 1μL.
Add the appropriate volumes of each reagent to labeled tubes.

Tube
Vol
DNA
10x Buff.
(ID :
)
Enzyme
(ID:
)
dH20
Total
Sample
1μL
1μL
1μL
7μL
10μL
Incubate 1 hour at 37oC
DNA Gel Electrophoresis
After the restriction digest has been completed, we need to demonstrate its presence in the solution
extracted from the bacteria. DNA gel electrophoresis is a procedure which sorts fragments of DNA into
bands of different sizes. If we know what size our plasmid is, we can identify the band which represents
that size by comparing it to a standard of known size (a “ladder”). Your tutors will tell you the size of the
plasmid which you are trying to isolate.
Size of Plasmid :
DNA electrophoresis is performed using a gel made out of agarose. The gel must be made fresh for each
test we perform. The running buffer, which is used in the electrophoresis tank, must also be made fresh.
The working running buffer (1X) is made by diluting the stock buffer (50X) 1 in 50 using distilled water.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
23
Dilution Factor Practice
You will need to prepare around 500mL of 1X TAE running buffer to fill the tank and make the
gel. Since you are provided with 50X TAE stock buffer, you will need to calculate how much
buffer to add to water to ensure a 1 in 50 dilution. Use the procedure below :
Total volume
= 500mL
1/50 of 500mL = 500 ÷ 50 = ____ mL
volume of 50X stock needed is ____ mL
Volume dH2O needed
= Total volume - Volume stock needed
= 500mL – ____ mL
= ____ ml
Dilute ____ mL stock in ____ mL of dH2O
Preparing the Gel (Your tutor will take you through this procedure)
The gel we will be using is 0.8% agarose. This means that we should add 0.8g of agarose to every
100mL. We do not need this much gel, however, so instead we will work on a total volume of
40mL. To adjust the masses involved, use the procedure overleaf:
New mass agarose = New volume
Old mass agarose
Old volume
New mass agarose = New volume x Old mass agarose
Old volume
= 40 x 0.8
100
=
g
Microwave the solution on HIGH for 2 minutes (for a small gel). Make sure that the agarose is
completely dissolved by swirling the heated mixture. Allow it to cool for 3 minutes.

TAKE CARE: The agarose solution is quite hot.
Use gloves and be careful not to spill any of
the solution.

TAKE CARE: Do not put a lid on the flask while
microwaving, otherwise the flask may
explode.
Wipe a plastic gel tray and comb with 70% ethanol and place in the electrophoresis tank so that
the rubber tubing forms a seal with the sides of the tank.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
24
Add 4µL of SYBR-Safe into the dissolved agarose and swirl to mix.
Pour the melted agarose into the gel tray. Place the comb into the right position and allow it to
set for approximately one hour (this can be done faster by placing the gel tray in the refrigerator.
Carefully remove the comb from the gel. Rotate the gel tray so that the wells are toward the
negative (black) terminals (the top of the tank, assuming that the electrodes are on the right
hand side). Cover the gel with 1X TAE running buffer.
Running the Gel
Samples are prepared by adding the appropriate amount of 6X loading dye to make it to 1X (i.e.
a 1 in 6 dilution). Since we are using the entire DNA digest to dilute the dye. The combined
volume of the digest solution and 6X stock dye must be 6 times the volume of the dye added.
if the volume of dye added is “x” :
x + Volume of DNA digest = 6x
 Use this calculation to complete the table below:
Volume of DNA

Volume of Dye
Total Volume
10µL of DNA Digest
5µL of DNA Ladder
Prepare loading solutions for your sample and DNA ladder.
Load all of the loading solutions into separate wells in the gel (loading the DNA ladder last into a
separate well on the left or right hand side of your gel). Use the table below to keep track of
where you have loaded each sample:

Sample
ID #1
Sample
ID #2
Loading End - Negative (Black) Electrode
Sample
Sample
Sample
Sample
ID #3
ID #4
ID #5
ID #6
Sample
ID #7
Sample
ID #8
Run the gel at 80V. There must be small bubbles rising from both ends of the electrophoresis
chamber. Check after 5 minutes to make sure the gel is running (i.e. the dye front has moved, is
relatively straight and has run the correct direction). Then allow the gel to run for the necessary
amount of time (about 1 hour however, check that the dye front has almost run through the gel).
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
25

TAKE CARE: While the electrophoresis tanks
are well insulated, they still feature high
voltages and conductive solutions.
Ensure
that the power pack is switched off and the
leads unplugged before opening the tank.
Switch off the power pack and take the gel to the illuminator. Take a photograph, print off and
glue into your workbook in the space below. Annotate the photograph using the ID table you
completed above, indicating bands of interest.
Pour away the buffer from the electrophoresis tank and rinse well with water. Rinse the gel tray
and comb as well.
(Affix your gel photograph here)
Interpreting Your Gel
Whenever we run a gel, we should always include a DNA “Ladder” which features
fragments of DNA of known size. This ladder serves as a reference point to indicate
the size of the DNA fragments in our sample. A map of the ladder we are using in this
exercise is provided in Figure 7.
Figure 7: Map of 1kb DNA Ladder
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
26
Appendices
APPENDIX A: SOME BASIC KNOWLEDGE
A1
DNA
Deoxyribonucleic acid (DNA) is a large molecule which stores the genetic information in
organisms. It is composed of two strands, arranged in a double helix form. Each strand is
composed of a chain of molecules called nucleotides, composed of a phosphate group, a five
carbon sugar (pentose) called deoxyribose and one of four different nitrogen containing bases.
NH 2
N
N
O
O
P
O-
N
O
N
O
5'
H
Phosphate
Group
O
H
H
3'
O
H
P
O
H
N
O-
NH
N
O
N
O
5'
H
H
Deoxyribose
H
3'
P
NH 2
NH 2
H
O
O
Nitrogenous
Bases
N
H
O-
O
O
N
O
5'
H
O
H
H
3'
O
P
O
H
HN
H
O-
O
O
N
O
5'
H
H
3'
OH
H
H
H
Figure 3 – The Structure of a Single Strand of DNA
Each nucleotide is connected to the next by way of covalent bonding between the phosphate
group of one nucleotide and the third carbon in the deoxyribose ring. This gives the DNA strand a
“direction” – from the 5’ (“five prime”) end to the 3’ (“three prime”) end. By convention, a DNA
sequence is always read from 5’ 3’ ends.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
27
DNA nucleotides contain one of four different nitrogenous bases:
Adenine
Guanine
Thymine
Cytosine
Each of these bases jut off the sugar-phosphate “backbone”. If the double helix of the DNA
molecule can be thought of as a “twisted ladder”, the sugar-phosphate backbones form the
“rails”, while the nitrogenous bases form the “rungs”.
The two strands of DNA are bound together by hydrogen bonding between the nucleotides.
Adenine always binds to thymine and guanine always binds to cytosine. This means that the two
strands of DNA are complementary. The complementary nature of DNA is allows it to be copied
and for genetic information to be passed on - each strand can act as a template for the
construction of its complementary strand.
The order of bases along a DNA strand is called the DNA sequence. It is the DNA sequence which
contains the information needed to create proteins through the processes of transcription and
translation.
Each strand of DNA is anti-parallel. This means that each strand runs in a different direction to
the other – as one travels down the DNA duplex, one strand runs from 5’
3’, while the other
runs 3’ 5’.
An animation of the structure of DNA can be found at:
http://www.johnkyrk.com/DNAanatomy.html
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
28
A2
DNA Replication
The structure of DNA allows it to carry out two vital functions for the cell :
 Encoding the information need to build and regulate the cell, and
 Transmission of this information from generation to generation
In order for the genetic information to be passed on, it must be copied. DNA replication occurs
during the S (synthesis) phase of the cell cycle. It only proceeds if the G1 checkpoint is passed,
which ensures that the chromosomes have properly segregated during mitosis.
In simple terms, DNA involves the separation of the two strands of the DNA molecule and the
construction of complementary strands for each one, using the A T, G C binding rules.
A GA AG CA TC CT AC GA GG G
A GA AG CA TC CT AC GA GG G
T C T G A G T C C
A GA AG CA TC CT AC GA GG G
T CA TG GA AC GT TC CA CG G
A G A C T C A G G
T CA TG GA AC GT TC CA CG G
Strands separate
T CA TG GA AC GT TC CA CG G
A new strand is built
for each, using the
original strand as a
template
Because the two new strands of DNA each contain one of the original parental strands, the
process of DNA replication is said to be semi-conservative (ie. half of the new DNA molecule are
strands “saved” from the parental molecule).
Naturally, the process of replication is a more complicated process than simply matching
nucleotide bases. Copying DNA involves the interplay of a series of enzymes and regulatory
processes, all kept in check by stringent error checking and repair mechanisms.
DNA replication begins when the enzyme helicase “unwinds” a small portion of the DNA helix,
separating the two strands. This point of separation is called the replication fork. The two
strands are kept separated by single stranded binding proteins (SSB) which bind onto each of
the strands. A group of enzymes called the DNA polymerases are responsible for creating the
new DNA strand, however they cannot start the new strand off, only extend the end of a preexisting strand. Therefore, before the DNA polymerases can start synthesizing the new strand,
the enzyme primase attaches a short (~60 nucleotides) sequence of RNA called a primer. The
DNA polymerases then extend this primer, moving along each strand from the 3’ end to the 5’
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
29
end and adding nucleotides to the 3’ hydroxyl group of the previous nucleotide base. The order
of nucleotides is retained by matching complementary nucleotides on the template strand.
DNA
Polymerase
Lagging strand
5
A
A
T
A
C
A
A
T
G
T
A
T
G
T
T
A
’
A
3
’
Okazaki Fragment
5
’
C
Helicase
G
3
’
5
T
T
T
A
C
A
A
T
G
A
A
T
G
T
T
A
C
T
’
3
’
DNA
Polymerase
Leading strand
It’s important to realize that the polymerases can only operate in one direction. This works out
for one of the DNA strands (the leading strand) – the polymerase moves along the strand in the
same direction as the replication fork. However the other strand (the lagging strand) runs in the
opposite direction. As a result the complementary strand to the lagging strand is made in short
sections called Okazaki fragments. These sections are then later joined together by the enzyme
DNA ligase.
Once the complementary strand of DNA has been synthesized, the primers are removed by the
enzyme RNAse H and the remaining gaps filled with lengths of DNA by DNA polymerase.
A
A
Some excellent animations of DNA replication can be found here :
This is a tutorial which takes you through the process step by step.
http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html
This animation is a computer generated movie showing what the process would look like on a
molecular level
G
http://www.youtube.com/watch?v=teV62zrm2P0
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
30
A3
PROTEINS
Proteins are large molecules composed of chains of amino
acids. Each amino acid is joined to the next one by peptide
bonds, which form between the acid (containing a carbon
atom) of one amino acid and the amino group (containing a
nitrogen atom) of the next. This means that all of the amino
acids are arranged in the same “direction” in the protein and
that one end of the protein can be designated the “carbon
end” (or C Terminus) and the other end the “nitrogen end” or (N terminus).
Because a protein consists of many amino acids joined by peptide bonds, the word “peptide” is
used to describe proteins – we may use “peptide chain” to describe the sequence of amino
acids, “oligopeptide” refers to a sequence of a few (e.g. a dozen or so) amino acids, while
“polypeptide” refers to sequences much longer and is equivalent to the term protein.
Because proteins are so large, we don’t often refer to their actual molecular mass. Instead we
use a unit called a Dalton, which is equivalent to a molecular mass of 100, or the average
molecular mass of an amino acid. Therefore, the size of a protein expressed in Daltons is
equivalent to how many amino acids it contains (e.g. a protein with a size of 244 Daltons is 244
amino acids long).
There are twenty different amino acids
found in nature. It is different combinations
of these amino acids which affect the
structure and function of the proteins.
Amino acids all have the same basic
structure, and only differ by the chemical
structure of a “side chain” which comes off
the central carbon atom. Amino acids are
sometimes referred to as “residues”.
You can find an excellent tutorial (including
a quiz) on amino acids at :
http://www.biology.arizona.edu/biochemis
try/problem_sets/aa/aa.html
Proteins can be thought of as having a
number of levels of structure – the primary,
secondary, tertiary and sometimes
quaternary structures. A video which briefly
describes each of these can be found at:
http://www.youtube.com/watch?v=lijQ3a8
yUYQ
Image from http://www.genome.gov/DIR/VIP/
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
31
Structures of the Twenty Amino Acids
Polar
Non-polar
“Special”
Aspartic Acid
(asp or D)
Glutamic Acid
(glu or E)
Lysine
(lys or K)
Arginine
(arg or R)
Histidine
(his or H)
Serine
(ser or S)
Threonine
(thr or T)
Glutamine
(gln or Q)
Asparagine
(asn or N)
Tyrosine
(tyr or Y)
Alanine
(ala or A)
Valine
(val or V)
Leucine
(leu or L)
Isoleucine
(ile or I)
Methionine
(met or M)
Phenylalanine
(phe or F)
Tryptophan
(trp or W)
Glycine
(gly or G)
Cysteine
(cys or C)
Proline
(pro or P)
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
32
The order of amino acids in a protein is called the primary structure. The peptide bond is a
covalent bond with partial double bond characteristics. This means that the molecules do not
rotate around the peptide bond and the bond remains rigid, giving some structure to the chain.
Because covalent bonds are quite strong, it takes a lot of energy to break up the primary
structure of a protein.
Once a chain of amino acids has been assembled by the ribosome, it tends to fold in on itself.
Nitrogen and oxygen atoms are strongly electronegative, which means that they will attract
hydrogen atoms bound to nearby atoms. The first hydrogen bonds to form tend to occur
between the atoms which make up the peptide bonds. This makes for tight structures within the
protein. When the chain coils up like a corkscrew, an α helix is formed. When the chain folds up
in parallel lines, a β pleated sheet is formed. Both of these components make up the secondary
structure of the protein. Whether a region of the peptide chain produces α helices or β pleated
sheet depends largely on the chemical nature of the amino acids which makes them up.
α Helix
β Pleated Sheet
(from Wikimedia Commons)
A protein then folds up to give further three-dimensional
structure, based on hydrogen bonds between the side
chains of its amino acids and their interaction with the
surrounding environment. If the side chain of an amino acid
is non-polar (hydrophobic or water repelling) it is pushed to
the inside of the protein molecule by the aqueous
environment inside the cell. Polar or charged side chains
tend to be found on the outside of the protein molecule.
This three-dimensional structure is the tertiary structure of
the protein. The tertiary structure determines the function
of the protein and how it interacts with other substances.
The tertiary structure of a protein,
showing α helices (red) and β pleated
sheets (gold)
(from Wikimedia Commons)
http://www.org.chemi.e.tuHydrogen bonds are quite weak compared to covalent bonds.(from:
It does
not take much to disturb a
muenchen.de/people/mh/Kdp/kdp.html)
hydrogen bond – gentle heating (e.g. above 60°C), changing the pH, the level of salts or the
presence of certain disrupting surfactants can all interfere with the hydrogen bonding which
holds the tertiary structure of proteins together. When this happens, we say that the protein has
been denatured. An example of this is when we heat egg white. The mostly transparent and
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
33
water soluble proteins which make up albumen are denatured by the heat, producing the hard,
insoluble and opaque substance we associate with cooked egg. Once a protein has been
denatured, it can no longer carry out its normal function – solubility changes and alterations
occur in the tertiary structure which means that the protein can no longer bind to other
substances or catalyse chemical processes.
Sometimes stronger bonds form between regions in the tertiary structure of proteins. Cysteine is
a sulphur containing amino acid. In proteins which contain a lot of this residue, nearby sulphur
atoms may form covalent disulphide bridges. These covalent bonds give a strength to the protein
not found in others. Tough proteins like keratin (found in fingernails and hair) contain a lot of
disulphide bridges.
The protein can be thought of as containing a number of distinct regions called domains. Each
domain either has a particular structure (e.g. a large number of α helices) or a distinct function in
the molecule. For example, while in most proteins, domains with a high proportion of non-polar
amino acids are forced to the inside of the molecule, transmembrane proteins tend to have
hydrophobic domains where these side chains are on the outside. This allows these proteins to
embed in and attach to the non-polar inner part of the cell membrane.
Some proteins, once assembled and folded, may join
together with other proteins to form larger molecules.
This is called the quaternary structure and is not found in
all proteins. If two or more identical subunits join
together, the results are called a homodimer,
homotrimer, homotetramer, etc. If the subunits are
different, they are called heteromers. For example, the
protein haemoglobin is a tetramer consisting of two α
chains and two β chains.
Other groups may also be attached to proteins to assist in
The quaternary structure of Haemoglobin,
its function. Lipids may incorporate to form lipoproteins showing the two pairs of subunits (red and
which may be associated with the cell membrane or gold) and the haem group (green)
(from: Wikimedia Commons)
involved in the transport of lipid substances like
cholesterol in the aqueous environment of the blood. Carbohydrates associate with proteins to
form glycoproteins, important as cell surface proteins involved in cellular recognition and in
structural components like cartilage.
Proteins with a transport or catalytic role may also incorporate metal ions encased in special
molecular cages. For example, each subunit in the haemoglobin molecule contains a carbon and
nitrogen containing cage called a haeme group which binds to the iron ions. This allows the
haemoglobin molecule to transport molecular oxygen throughout the body in the blood. Such
proteins are called metalloproteins.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
34
Protein Biochemistry Links
Folding@Home – a distributed computer project aimed at investigating how proteins fold.
Download the software and help protein scientists in their research.
http://folding.stanford.edu/
Folding@Home’s guide to Proteins – excellent summary from amino acids to protein folding.
Includes some great animations.
http://www.stanford.edu/group/pandegroup/folding/education/protein.html
Introduction to Protein Structure – a presentation by Dr Frank Gorga.
http://webhost.bridgew.edu/fgorga/proteins/default.htm
Interactive Concepts in Biochemistry – a wide range of molecular biology related interactive
activities, including an amino acid identification game.
http://www.wiley.com/legacy/college/boyer/0470003790/animations/animations.htm
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
35
A4
TRANSCRIPTION AND TRANSLATION
Genetic information is stored in the sequence of nucleotide bases in the DNA molecule. When
this information is presented in an organism, we say that it has been expressed. The evidence of
expression is the presence of a protein, made using the instructions present in the genetic code.
The first step in the expression of a protein is transcription. This is the construction of a
specialised nucleic acid called messenger RNA (mRNA) based on one strand of the DNA molecule.
RNA differs from DNA in that it is single stranded (although it may double over on itself in
regions), it contains the pentose ribose instead of deoxyribose, and it contains the nucleotide
base uracil rather than thymine.
RNA synthesis is catalysed by enzymes called
C RNA polymerases (in eukaryotes, RNA polymerase
II). RNA polymerase binds to a sequence of nucleotides on the DNA molecule before the gene to
be transcribed called the promoter. Promoters guide the RNA polymerase where to begin
transcription, which direction to start transcription, and which strand of DNA to transcribe.
Promoters can bind with varying strengths, which is one reason why some proteins are
expressed more than others (i.e. their promoters bind more strongly).
The RNA polymerase (along with some “helper” proteins) then unwinds a small portion of the
DNA duplex. Using one strand as a template, it assembles a strand of mRNA from
ribonucleotides.
RNA
Polymerase
5’
3’
C
G
DNA
G
C
A
T
T
A
C
G
C
G
A
T
A
A
T
T
A
G
C
C
G
C
G
3’
5’
C
mRNA
G
A
U
G
G
T
A
A
U
T
A
3’ Ribonucleotides
added to this end
5’
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
36
One strand of DNA is designated the “coding strand”, while the other is the “template strand”.
mRNA is constructed using the template strand, with the enzyme moving down this strand from
3’ to 5’. Ribonucleotides are added to the 3’ end.
As RNA polymerase moves along the DNA molecule, the separate strands join back together
again.
When the RNA polymerase reaches a termination sequence, the enzyme drops off the DNA
molecule and the mRNA molecule finds its way to a ribosome for the process of translation. In
prokaryotes, where there is no nucleus, translation occurs immediately. In eukaryotes, the
mRNA is modified so that it can make it out of the nucleus and into the cytoplasm. The 5’ base is
modified (“capping”), a long stretch of adenosines are added to the 3’ end (“poly-A tail” and
sections of non-coding RNA (introns) are removed and the remaining RNA (exons) joined back
together.
The mRNA attaches to the ribosome, either through a binding sequence on the mRNA (in
prokaryotes) or via the 5’ cap (in eukaryotes). The ribosome then moves down the mRNA until it
finds the start sequence (AUG).
Floating around the ribosome are various tRNA (transfer RNA) molecules. Each of these has a
specific sequence of three bases which is complementary to bases in the mRNA. The sequences
on the tRNA are called “anti-codons” which bind to their complementary “codons” on the mRNA.
For each anti-codon, a different amino acid is also bound to the tRNA.
e.g.
The start codon is AUG. The anti-codon for this is UAC – this sequence is found on a tRNA
which only binds to the amino acid methionine.
There are 20 different amino acids and 64 different combinations of A, U, G and C. This allows for
a certain level of redundancy (i.e. one amino acid may be coded for by more than one codon) as
well as three “stop” codons. The list of which codons code for each amino acid is known as the
genetic code.
st
1
position
(5’ end)
U
C
A
G
2
U
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
lle
lle
lle
Met
Val
Val
Val
Val
nd
C
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
rd
position
A
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
G
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AAG
GGU
GGC
GGA
GGG
Cys
Cys
Stop
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
3
position
(3’ end)
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
37
The ribosome holds the mRNA in place until the tRNA with the corresponding anti-codon binds
onto the codon on the mRNA. Then the tRNA containing the anti-codon for the next codon binds
next to it. The amino acids abound to each of these tRNA molecules are brought close together
and a peptide bond formed between them by the enzyme peptidyl transferase.
The ribosome then moves down the mRNA to the next codon and the process is repeated. New
amino acids are added to the C-Terminus of the growing peptide chain. As amino acids are
joined, they separate from the tRNA and the peptide chain is freed.
When the ribosome reaches a stop codon, no tRNA with an amino acid exists to bind, so the
peptide chain separates and goes on to fold into its secondary and tertiary structures.
It is important to understand the relationship between the DNA sequence and the sequence of
amino acids in the peptide chain. The coding strand of the DNA, read from 5’ to 3’ gives the
sequence of bases which codes for the protein (substituting “U”s for “T”s). In other words, the
coding sequence, written 5’ to 3’, gives the amino acid sequence from N terminus to C terminus.
An excellent animation explaining this process can be found at:
http://www.wiley.com/legacy/college/boyer/0470003790/animations/translation/translation.htm
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
38
APPENDIX B: CELL AND MOLECULAR BIOLOGY TECHNIQUES
B1
DNA RESTRICTION
Restriction enzymes cut the DNA at a particular point, determined by a nucleotide sequence (e.g.
the EcoR1 restriction enzyme cuts the DNA wherever it finds the sequence GAATTC)
Some enzymes cut the DNA molecule unevenly i.e. they cut one strand of the double helix at one
point, but then cut the other strand “downstream” of this point. EcoR1 is an example of one of
these restriction enzymes. When it cuts the DNA molecule, it leaves two single stranded ends of
TTAA. These are called “sticky ends” because they are complementary to each other and will
bind again if brought close to each other
If the two sticky ends are bound to each other, another enzyme called DNA Ligase can reform
the DNA molecule by rejoining the phosphate/sugar backbones.
These restriction enzymes are used to insert fragments of DNA into other DNA molecules – if the
DNA is cut with sticky ends and a fragment with complementary sticky ends is introduced, the
fragment may be inserted into the gap in the DNA. This is one of a ways that we introduce new
DNA in genetic recombination.
Other restriction enzymes simply cut the DNA up into smaller fragments – every time the
restriction enzyme finds its binding site on the DNA, it cuts the molecule. Because individuals
with a different nucleotide sequence will have different frequencies of these binding sequences,
each different sequence treated with a particular restriction enzyme will yield a different pattern
of small and large fragments which can be demonstrated using gel electrophoresis. This is the
basis of techniques such as DNA profiling (aka DNA “fingerprinting).
Visit: http://www.dnalc.org/ddnalc/resources/animations.html and select the “DNA Restriction”
animation.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
39
B2
AGAROSE GEL ELECTROPHORESIS FOR DNA
Electrophoresis is a technique used to separate large molecules like proteins and nucleic acids
based on their size. It relies on the principle that larger molecules take longer to migrate through
a porous medium than smaller molecules.
Nucleic acids are composed of chains of nucleotides. The “backbone” of the nucleic acid
structure is a repeating chain of phosphate groups and pentose sugars. At certain pH values, the
oxygen atoms in the phosphate groups ionise, giving the molecule an overall negative charge. If
exposed to an electric field, these molecules are attracted to the positive terminal.
DNA electrophoresis occurs through a gel composed of agarose, a compound derived from
seaweed. This is immersed in a solution of a buffer (a substance which maintains a constant pH)
which has the dual role of conducting electricity and ensuring that the DNA molecules are at a
consistent pH to ensure ionisation.
The gel is then placed in an electrophoresis tank loaded with buffer. Solutions containing the
DNA are mixed with a blue dye (which travels before all of the DNA and shows us how far the
samples have traveled) and loaded into wells in the gel. A power supply is connected, with the
negative terminal to the loading end. The process is complete when the dye front has almost
reached the positive end of the gel.
Gels are normally made containing a compound called Ethidium Bromide. This substance binds
to DNA and fluoresces when exposed to ultraviolet light. Once the gel has run, it is photographed
under UV light. DNA fragments of a similar size run together as “bands”. Smaller fragments can
penetrate further through the gel than larger fragments in the same time. This means that bands
of smaller DNA fragments will be found closer to the positive terminal, while larger fragments
will be found closer to the loading wells.
One well in a gel is always left for markers. This is a mixture of DNA fragments of known size
which separate out into a ladder. We can estimate the size of the fragments we are investigating
by comparing how far they have migrated with the migration of fragments of known size.
Visit: http://www.dnalc.org/ddnalc/resources/animations.html
Electrophoresis” animation.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
and
select
the
“Gel
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
40
B3
IMMUNOFLUORESCENCE
Immunofluorescence is a technique used to demonstrate structures and substances in cells and
tissues. It relies on the tendency of antibodies to bind specifically to certain materials.
An antibody is a protein produced by cells
in the immune system. It is made up of a
number of subunits consisting of separate
polypeptide chains. There are two major
types of subunit – the heavy and light
chains, and each single antibody has two
light chains and two heavy chains.
Variable Regions
Antibodies can be thought of as having a
shape like a capital letter Y. Their
functionality is determined by the variable
regions, found at the ends of each of the
branches of the Y. The variable regions are
made up of sections of the heavy and light
chains.
Light Chain
Heavy Chain
Antibodies work by binding to other
substances called antigens through the
variable region. The region of an antigen
to which an antibody binds is called the
epitope.
Generalised Antibody Structure
Each antibody produced has a variable region which specifically binds onto the epitope of a
single antigen. This is what gives antibodies their specificity.
In the body, antibodies are produced as part of the humoral immune response. When cells in the
body come into contact with an antigen, they “instruct” specialised B lymphocytes to make
antibodies which can bind to that antigen – each line of cells produces just one type of antibody.
Once an antibody has been produced, it will bind to just that antigen. If the antigen forms part of
a disease agent like a bacterium, parasite or virus, the antibody may assist in the body’s immune
response by binding to that antigen and :
o
interfering with the agent’s ability to cause damage to the body (eg. antibodies binding to
external proteins on a virus prevent those proteins from interacting with receptors on the
outside of a host cell, preventing the cell from taking up the virus)
o
causing a phagocytic immune cell to engulf and destroy the disease agent
o
causing the disease agent to clump together and not be able to multiply or cause damage to
the body
o
initiating destructive immune processes (eg. complement activation)
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
41
In addition to their role in immunity, antibodies are widely used in biological research and
diagnosis because of their specificity. Antibodies can be mass produced in test animals by
exposing those animals to antigens of interest and harvesting the cell lines which produce the
antibody we are interested in. If a cell line produces just one antibody, the antibodies produced
are called monoclonal antibodies. Polyclonal antibodies have a number of different binding
affinities and come from multiple cell lines.
Immunofluorescence involves the use of antibodies which bind to cell components of interest,
bound to a chemical substance which fluoresces under ultraviolet light (a flurophore). There are
two main types of immunofluorescence :
o
Direct Immunofluorescence involves the use of a single labeled antibody. Because there is
only one labeled antibody per antigen site, direct immunofluorescence gives a lower signal
and is therefore less sensitive.
o
Indirect Immunoflourescence involves the use of an unlabelled primary antibody which
binds to the target antigen, and then a labeled secondary antibody which binds to the
primary antibody. Because there may be multiple sites on the primary antibody for the
secondary antibody to bind, the signal can be much stronger and indirect
immunofluorescence is generally more sensitive than direct immunofluorescence.
The following video shows the process of indirect immunofluorescence :
http://www.youtube.com/watch?v=OH2GFeaGV6w
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
42
Direct Immunofluorescence
Antigen Present
Antigen Absent
Treatment with labeled antibody
Antigen Present
Antigen Absent
Unbound antibody washed away
UV Light
Antigen Present
UV Light
Antigen Absent
Fluorescence observed where antigen is located
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
43
Indirect Immunofluorescence
Antigen Present
Antigen Absent
Treatment with unlabelled primary antibody
Antigen Present
Antigen Absent
Unbound antibody washed away
Antigen Present
Antigen Absent
Treatment with unlabelled secondary antibody
(continued over)
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
44
Indirect Immunofluorescence (continued)
Antigen Present
Antigen Absent
Unbound secondary antibody washed away
UV Light
UV Light
Antigen Present
Antigen Absent
Fluorescence observed wherever antigen is located
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
45
APPENDIX C: STANDARD OPERATING PROCEDURES
C1
USING A MICROPIPETTE
When scientists need to accurately and precisely deliver smaller volumes of a liquid, they use a pipette –
a calibrated glass tube into which the liquid is drawn and then released. Glass and plastic pipettes have
been mainstays of chemistry and biology laboratories for decades, and they can be relied upon to
dispense volumes down to 0.1mL.
Molecular biologists frequently use much smaller volumes of liquids in their work, even getting down to
0.1µL (that’s one ten thousandth of a millilitre, or one ten millionth of a litre!). For such small volumes,
they need to use a micropipette.
Plunger
Tip Eject
Button
Volume
Adjustment
Volume
Readout
0
5
0
Tip Eject
Shaft
Micropipettes are called a lot of different names,
most of which are based on the companies which
manufacture. For example, you might hear them
called “Gilsons”, as a large number of these
devices used in laboratories are made by this
company. Regardless of the manufacturer,
micropipettes operate on the same principle: a
plunger is depressed by the thumb and as it is
released, liquid is drawn into a disposable plastic
tip. When the plunger is pressed again, the liquid is
dispensed.
The tips are an important part of the micropipette
and allow the same device to be used for different
samples (so long as you change your tip between
samples) without washing. They come in a number
of different sizes and colours, depending on the
micropipette they are used with, and the volume
to be dispensed.
Tip
Attachment
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
46
The most commonly used tips are:
Large Blue – 200-1000µL
Small Yellow – 2-200µL
Small White - <2µL
They are loaded into tip boxes which are often sterilised to prevent contamination. For this reason tip
boxes should be kept closed if they are not in use. Tips are loaded onto the end of the micropipette by
pushing the end of the device into the tip and giving two sharp taps. Once used, tips are ejected into a
sharps disposal bin using the tip eject button. Never touch the tip with your fingers, as this poses a
contamination risk.
The plunger can rest in any one of three positions:
Position 1 is where the
pipette is at rest
Position 2 is reached by pushing
down on the plunger until
resistance is met
Position 3 is reached by pushing
down from Position 2
Each of these positions plays an important part in the proper use of the micropipette.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
47
To Draw Up Liquid:
Hold the micropipette with the thumb resting on the plunger and the fingers curled around the
upper body.
Push down with the Keeping the plunger at the second
thumb until Position 2 is position, place the tip attached to
reached.
the end of the micropipette
beneath the surface of the liquid to
be drawn up. Try not to push right
to the bottom (especially if you are
removing supernatant from a
centrifuged pellet), but ensure that
the tip is far enough below the
surface of the liquid that no air is
drawn up.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
Steadily release pressure on
the plunger and allow it to
return to Position 1. Do this
carefully, particularly with
large volumes, as the liquid
may shoot up into the tip and
the body of the micropipette.
If bubbles appear in the tip,
return the liquid to the
container by pushing down to
Position 3 and start again (you
may need to change to a dry
tip).
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
48
To Dispense Liquid:
Hold the micropipette so that the end of the tip containing tip is inside the vessel you want to
deliver it to. When delivering smaller volumes into another liquid, you may need to put the end
of the tip beneath the surface of the liquid (remember to change the tip afterwards if you do this
to save contaminating stock). For smaller volumes you may also need to hold the tip against the
side of the container.
Push the plunger down to
Position 2. If you wish to mix
two liquids together or
resuspend a centrifuged
pellet, release to Position 1
and push to Position 2 a few
times to draw up and expel
the mixed liquids
To remove the last drop of
liquid from the tip, push down
to Position 3. If delivering into
a liquid, remove the tip from
the liquid before releasing the
plunger
Release the plunger and allow
it to return to Position 1
Changing the Volume:
Some micropipettes deliver fixed volumes, however the majority are adjustable. Each brand uses a
slightly different method to do this – Gilsons have an adjustable wheel, others have a locking mechanism
and turning the plunger adjusts the volume. All have a readout which tells you how much is being
delivered and a range of volumes which can be dispensed. Trying to dipense less than the lower value of
the range will result in inaccurate measurements. Trying to dispense over the upper range will
completely fill the tip and allow liquid to enter the body of the pipette. Do not overwind the volume
adjustment, as this affects the calibration of the micropipette.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
49
The way to interpret the readout depends on the micropipette used:
0
P
1000
3
5
0
P
200
9
5
0
P
20
2
5
0
P2
5
0
In a 200-1000µL micropipette (e.g. a Gilson
P1000) the first red digit is thousands of µL (it
should never go past 1), the middle digit is
hundreds, while the third is tens. Therefore
1000µL would read as 100, while 350µL would
read as 035.
In a 20-200µL micropipette (e.g. a Gilson P200)
the first digit is hundreds of µL (it should never
go past 2), the second is tens and the third is
units. Therefore, 200µL would read as 200,
while 95µL would read as 095.
In a 2-20µL micropipette (e.g. a Gilson P20) the
first digit is tens of µL (it should never go past
2), the second is units and the third red digit is
tenths. Therefore 20µL would read as 200,
while 2.5µL would read a 025.
In a 0.2-2µL micropipette (e.g. a Gilson P2) the
first digit is units of µL (it should never go past
2), the second red digit is tenths and the third
red digit is hundredths. Therefore, 2µL would
read as 200, while 0.5µL would read as 050.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
50
C2
USING A BENCH CENTRIFUGE
If a suspension of materials containing different densities are allowed to sit, gravity will separate them;
with the materials of the highest density sinking to the bottom and the lower densities floating on top.
This is the reason why air floats above water and why cells in a blood sample will sink towards the
bottom of the tube. In laboratories, we cannot afford to wait around for gravity to take its course, so we
use centrifuges. Centrifuges are devices which use high rotational speeds to increase the rate at which
materials settle out according to density.
In molecular biology, centrifuges are used for a variety of purposes – spinning down small volumes of
liquids which may have collected on the sides of a vessel, separating and washing cells and forcing liquids
through separation columns as in gel purification. At high enough rotational speeds (ultracentrifugation),
we can even separate cell components and organelles, or even macromolecules based on their size.
The most commonly used bench centrifuges are used to separate
small volumes (~1.5mL). These are also called microcentrifuges or
microfuges. Centrifuges need to use specialised vessels, called
centrifuge tubes. Sometimes they are given the names of the most
prominent manufacturers of these vessels – you may often hear
microfuge tubes called “Eppendorfs” after the manufacturer,
although not all microfuge tubes are made by this company.
When using any centrifuge, the most important concept to keep in
mind is that of balance. The tubes are spun at extremely high
velocities (up to 13,000rpm for a simple microfuge), so any
irregularity in mass between tubes can set up instability in the system. At low speeds, this can cause
wobbling and a loud noise, while at high speeds, it can cause catastrophic failure with serious impacts on
safety. In both cases, irreparable damage can be caused to the centrifuge.
In a bench centrifuge, tubes are placed into a solid rotor – the centrifuges we will be using have spaces
for 24 x 1.5mL microfuge tubes.
1
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
51
When placing the tubes into the rotor, keep the following points in mind:
Make sure that tubes are placed into the rotor opposite each other. You can ensure this by
imagining a line passing between the two tubes. If the line passes through the centre of the
rotor, the centrifuge is balanced.
1
 Tubes are balanced
1
 Tubes are not balanced
Even if the tubes are in the correct position in the rotor, they need to be of equal mass (i.e. they
need to contain the same volume of liquid). If your tubes have unequal volumes or you have an
odd number of tubes, make sure that you include a balance tube containing the correct volume
of liquid.
 Tubes are balanced
 Tubes are not balanced
Make sure that the lid is attached to the rotor before you spin. This also reduces the risk posed
by aerosols formed when liquids are spun at high speeds.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
52
C3
USING A HAEMOCYTOMETER
In order to provide a standard environment for experimental studies, tissue culture techniques require a
consistent number of cells to be cultured. Too few cells may result in insufficient material, while too
many cells may challenge the cells in such a away to affect the outcome of the experiment.
Many laboratories use automated cell counters like Coulter counters or flow cytometers when
processing large numbers of samples. However, when checking the numbers of cells for serial dilutions
or setting up cultures, it is sometimes easier to return to traditional methods of cell counting.
The haemocytometer is a modified and calibrated microscope slide designed to allow operators to
quickly estimate the concentration of cells in a sample. The cells present in a known volume are counted
and then this value converted to a number per mL. The name refers to its original use in counting blood
cells – blood was diluted to a point where the cells could be reliably counted and this was factored up
based on the dilution.
Grid
Raised Glass
“Rails”
Counting
Chamber
Coverslip
The cell suspension is introduced to a space of known depth (0.1mm) beneath a coverslip and counted
within a grid of area 1mm2 (see over). This gives the number of cells per 0.1mm3 (or 0.1µL). Multiplying
by 10 gives the number of cells per µL and multiplying by 10,000 gives the number of cells per mL.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
53
Close-up of grid with cells
In the above example, there are 24 cells within the grid (do not count any cells outside the grid).
24 cells per 0.1 µL
= 240 cells per µL
= 2.4 x 105 cells per mL
In this example to deliver 1 x 106 cells, one would have to measure out 4mL of cell culture suspension.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
54
If the cell count is too high, try counting the cells in a fraction of the larger squares.
E.g.
If you counted 5 of the larger squares and arrived at a number of 100 cells
100 x 5 (there are 25 larger squares in total)
= 500 cells per 0.1µL
= 5000 cells per µL
= 5 x 106 cells per mL
In this example, you would have to deliver 200µL of cell suspension to provide 1 x 106
cells
Setting up the Haemocytometer:
Wet the raised glass rails with the tip of a moistened finger
Carefully slide the coverslip over the raised glass rails
Draw up 10µL and deliver into the gap between the coverslip and the counting chamber
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
55
C4
USING A BIOLOGICAL SAFETY CABINET
Biological safety cabinets are used wherever we want to limit contamination, such as when we are
working with pathogenic (disease-causing) organisms or when contamination from outside will seriously
compromise our work (e.g. with cell culture).
Unlike Laminar Flow Cabinets (which drawn in air and expel it unfiltered) or Fume Cabinets (which draw
in air and expel it outside the room), Biological Safety Cabinets circulate air through a series of high
quality filters. Because we want to have a non-contaminating environment inside the cabinet, we need
to follow a set procedure whenever we use them.
When Starting Up:
Remove the metal sash from the front of the cabinet, stacking it on the floor next to it
Turn the cabinet on – you should hear a rush of air and the light should turn on
Spray the work area with 70% ethanol and wipe with a paper towel
When Using:
Gather all of the items you will be using
All items which enter the cabinet must be decontaminated by spraying with 70% ethanol and
wiping with paper towel before being placed inside
To prevent contamination, briefly spray and rub your gloved hands with 70% ethanol before
placing then in the cabinet
Perform all work with your hands inside the cabinet
When Finished:
Remove all gear from inside the cabinet
Unscrew the waste jar from the vacuum line and tip the contents down the sink with a lot of
water following it. Rinse and place a squirt of iodine decontaminant into the jar. Replace on the
vacuum line
Spray the work area with iodine decontaminant and wipe with a paper towel
Spray the work area with water and wipe with a paper towel
Spray the work area with 70% ethanol and wipe with a paper towel
Turn the cabinet off and replace the metal sash
Press the “UV” button – you should see the UV light turn on. It will remain on for 20 minutes (Do
not use the cabinet while the UV light is on)
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
56
C5
FLUORESCENCE MICROSCOPY
A fluorescence microscope (which uses incident ultraviolet light rather than light from the visible
spectrum) is used to detect, localize and quantitate the amount of fluorescence in a specimen, and uses
this to indicate the level of the labeled agent we are looking for.

TAKE CARE: The fluorescence microscope uses
ultraviolet light. This can be damaging to your
eyes and skin. Always ensure that the
appropriate shields and filters are in place
when using the microscope
Carl Zeiss WIDEFIELD Fluorescence Microscope

REMEMBER: The high power objective uses oil to
cut down interference in air. DO NOT use oil on
the dry objective lenses
DRY objectives: 5x, 10x, 20x, 40x
OIL objectives: 63x
A
4
3
1
6
5
2
B
Turn on the computer , lamp power source , lamp  (ignite the lamp by holding down the
ignition button  until the orange light stays lit), microscope , and camera .
Open up the “SPOT Advanced” program on the computer desktop.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
57
Observing specimens and taking photographs using a fluorescence microscope is not quite the same as
using a standard light microscope. Fluorophores are excited by being irradiated with a narrow range of
wavelengths (as opposed to the large range of wavelengths in visible light). The narrow range of
excitation wavelengths is achieved by passing the light through a filter. This means that usually, only one
fluorophore can be visualized at any time. Therefore, if a specimen has been treated with a range of
flurophores, these must be observed and photographed separately.
The light emitted by the fluorophores is often of a low intensity. Colour cameras are usually not sensitive
enough to detect this light, so the camera takes images in grey scale. These images must be given a false
colour to differentiate each fluorophore.
Therefore, the general procedure when taking photographs using a fluorescence microscope is :
Select a field
Select a filter
Take a photograph
Select the next filter without changing the field
Take a photograph
Select the next filter without changing the field
Take a photograph
Give each of the images taken a false colour based on the flurophore
Merge the three colourised images together to create a three colour image
Taking the Images
For the fluorescence microscope in the SPARQ-ed laboratory, use the following procedure :
Place the slide on the microscope stage and position so that the specimen is directly over the
lamp.
Make sure that the light path selector A is set at position “A” (100% to eyepiece)
Set the sliding filter selector B to “UV” (this is the setting for DAPI)
Focus on a field with good coverage of DAPI stained nuclei (they should appear pale blue)
Set the light path selector to position “C” (100% to camera at side)
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
58
In SPOT Advanced, select “Camera”, “Live Image”
An image of the field should appear in the live image window. Adjust focus if necessary. If the
image is too dim, increase the exposure time (expressed in milliseconds – 1 second = 1000ms) via
the “Controls” button.
When you are happy with the image, click “Snap”. The image will appear on the screen.
It is a good idea to retain your raw images – sometimes a strong overlapping signal may drown
out a weaker one. Click “File”, “Save As” and select a unique filename that contains the word
“Blue” to indicate that this is the blue DAPI channel. Save the file as a .jpeg and reduce this
image down if required.
Change the filter slider to the green “B2” filter. Turn the light path selector back to “A” and make
sure that the cells are in focus DO NOT CHANGE THE FIELD OF VIEW.
Change the light path selector to “C” and click on “Camera”, “Live Image”. Adjust the exposure if
necessary as before
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
59
When you are happy with the image, click “Snap”. The image will appear on the screen.
Click “File”, “Save As” and select a unique filename that contains the word “Green” to indicate
that this is the green channel. Save the file as a .jpeg and reduce this image down if required.
Change the filter slider to the red “↕” or “G”filter (depending on the Flurophore ↕ is intended
for use with Texas Red, G is intended for use with TRITC). Turn the light path selector back to “A”
and make sure that the cells are in focus DO NOT CHANGE THE FIELD OF VIEW.
Change the light path selector to “C” and click on “Camera”, “Live Image”. Adjust the exposure if
necessary as before.
When you are happy with the image, click “Snap”. The image will appear on the screen.
Click “File”, “Save As” and select a unique filename that contains the word “Green” to indicate
that this is the green channel. Save the file as a .jpeg and reduce this image down if required.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
60
Colourising and Merging the Images
Call up the “Blue” image
Select “Edit”, “Select Palette”, “Blue”, “OK”. The image should take on a blue tone
Call up the “Green” image
Select “Edit”, “Select Palette”, “Green”, “OK”. The image should take on a green tone
Call up the “Red” image
Select “Edit”, “Select Palette”, “Red”, “OK”. The image should take on a red tone
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
61
Select “Edit”, “Merge Images”.
Ensure that all files are included (check all boxes).
Click “OK”
The merged, three-colour image should be presented on the screen – make sure that you save it
under a new filename.
Shutdown Procedure
Clean oil off objectives using lens tissue. For heavy contamination, use cotton bud immersed in
100% EtOH and gently clean objective, followed by Lens tissue wipe.
Ensure Mercury lamp has been on for 15 minutes before switching it off.
Turn off Computer and wait for full shut down
Turn off all switches
Cover microscope (leave hot lamp at the back uncovered)
Turn off wall switches
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
62
APPENDIX D: GLOSSARY OF TERMS
Actin – a protein which plays a major role in the cytoskeleton and cellular movement.
Adaptor Protein – a protein which transmits the signal from a receptor to a signal transduction cascade.
Allele – a variation of a particular gene.
Aliquot – to deliver a measured sample of a liquid
Amino Acid General Structure
Amino Acid – a small organic molecule which has a component
which acts as a base (an amine group –NH2) and one which acts as an
acid (carboxyl group –COOH). Amino acids are the building blocks of
proteins. Hanging off the central carbon atom is a side chain (often
given the abbreviation R) which differentiates one amino acid from
another. There are approximately 20 different amino acids which are
used to make proteins. The amino acids are normally written as
either a standard three letter or single letter code. See Appendix A3
for a list of amino acid structures and their three and single letter
codes.
Analogue – a compound with a chemical structure similar to another compound.
Androgen – hormones responsible for the development and maintenance of male characteristics.
Androgen Ablative Therapy – a type of therapy for prostate cancer which involves limiting the effect
androgens like testosterone have on the growth and progression of cancer cells.
Antibody – a specialised protein molecule produced as part of the immune response which binds
specifically to other molecules.
Aspirate – to remove the liquid from a sample using a vacuum line. Aspirated materials are usually
disposed of.
Autocrine – referring to a hormone system in which the hormone affects the cells which produced it.
Autophosphorylation – the process of an enzyme attaching a phosphate group to itself or to identical
enzymes,
Band – region of a gel in which proteins or DNA fragments of a particular size are concentrated after
electrophoresis.
Buffer – a solution of chemicals which resists the change of pH which would normally occur when an acid
or base is added.
Cancer – a family of conditions characterised by uncontrolled cell growth, infiltration into neighbouring
tissues and occasionally spread to other tissues in the body.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
63
Carbohydrate – a family of biomolecules based around sugars.
Castration – removal of the testes. As these organs are the primary producers of testosterone in males,
castration is used as an androgen ablative therapy.
Catalytic Domain – the region of an enzyme which assists in chemical reactions.
Caveolae – small, flask-like invaginations of the cell membrane involved in signal transduction.
Caveolins – a family of proteins, of which Caveolin-1 is principally responsible for the formation of
caveolae.
Cavin-1 – a protein which binds onto Caveolin-1 which is essential for the formation of caveolae. Also
known as PTRF (Polymerase 1 and Transcription Release Factor).
Cell – membrane bound bodies which form the basis of all living things.
Cell Membrane – the outer boundary of the cell. It is composed of a double layer of phospholipids
molecules arranged so that the water soluble ends face outwards while the water insoluble fatty ends
face inwards. Embedded in this bilayer are various proteins involved in recognition of other substances
and transfer of materials across the membrane.
The Structure of the Cell Membrane
Centrifuge – a piece of laboratory equipment which separates components in a liquid medium based on
their relative densities. Samples are loaded into tubes and spun at high speed. Denser materials sink to
the bottom, while less dense materials float to the top.
Chromosome – a length of DNA containing a long sequence of genes.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
64
Cholesterol – a steroid derivative which plays an important role in the structure of the cell membrane, as
well as being a pre-cursor to steroid hormones and other compounds in the body.
Codon – a sequence of three nucleotide bases which encodes a particular amino acid in a peptide
sequence.
Comb – a plastic template used to cast loading wells into
electrophoresis gels.
Competent Cells – bacteria capable of taking in DNA from their
environment. Commercially, competent cells are usually defined as
those capable of 106-108 transformations per μg of transforming DNA.
Conformation – the 3 dimensional shape of a large molecule
like DNA or a protein.
Electrophoresis Combs
Conjugate – a large molecule composed of two different molecules bound together.
Construct – an artificial length of DNA composed of DNA from different sources. For example, when
plasmids are used as vectors, they often consist of the plasmids itself (often of bacterial origin), the gene
to be inserted into the test cell, sequences which restriction enzymes target and genes for selection
purposes (e.g. genes for antibiotic resistance).
C-Terminus – the end of the protein chain which has the carboxyl group exposed.
Cuvette – a small container made of glass, plastic or quartz crystal used to hold the substances tested in
spectrophotometry.
Cytoplasm – the jelly-like substance outside the nucleus and bound by the cell membrane. It consists of
the cytosol (cell liquid) and the organelles and other cellular components.
Cytoskeleton – a network of structural proteins which supports the cell and facilitates movement of
components within it.
Development – the change in function of a cell, tissue, organ or organism as it ages.
Differentiation – the formation of specialised cells from generic pre-cursor or stem cells.
Dimer – a molecule consisting of two smaller subunits.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
65
DNA – deoxyribonucleic acid. DNA is composed of two antiparallel chains of
nucleotides arranged in a double helix conformation. DNA resembles a
twisted ladder, with the “rails” consisting of alternating phosphate groups
and the 5-carbon sugar deoxyribose, and the “rungs” composed of pairs of
nitrogenous bases joined by hydrogen bonding. It is capable of making copies
of itself (with the aid of enzymes such as DNA polymerases) and the order of
its bases stores the information needed to manufacture proteins.
Domain – a region within a protein molecule which has a particular function.
Downstream – sequences of amino acids in proteins are always written from
the N-terminus to the C-terminus. If a sequence is said to be inserted
“downstream” from a target gene, this means that it is attached after the Cterminus.
E. coli – Escherichia coli – a gut bacterium sometimes called the “workhorse
of molecular biology” because it is so commonly used in these laboratories.
Electrophoresis – a process by which an electric field is used to separate large
molecules (e.g. DNA or proteins) on the basis of their size through a gel.
Endocytosis – the process by which cells take in substances from the external
environment by surrounding them with pockets of the cell membrane.
Endosome – an intracellular membrane-bound body formed as the result of endocytosis.
Enzyme – a protein molecule which catalyses (helps along) a chemical reaction by lowering its activation
energy. Enzymes do this by bringing molecules close together to join them together, or by undergoing a
conformational change which breaks a bond.
Epidermal Growth Factor (EGF) – a small peptide which triggers cell growth and proliferation.
Epidermal Growth Factor Receptor (EGFR) – a type of tyrosine kinase receptor which binds to EGF.
Epithelium – a tissue which lines a body surface.
ERK (Extracellular Signal-regulated Kinase) – an alternative name for MAPK1 and MAPK2. ERK is
sometimes used in place of MAPK.
Eukaryote – an organism whose cells containing membrane-bound organelles (eg. protists, fungi, plants
and animals)
Expression – the production of a protein under the instruction of a gene.
Fluorescence – the emission of visible light by a substance after it has been excited by ultraviolet light.
Frameshift – a mutation which causes a change in the reading frame of a sequence of nucleotides.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
66
Gel – a semi-solid substance made by joining together smaller molecules
(monomers) into larger molecules (polymers) in the presence of water.
Gels are used as a supporting medium for electrophoresis. Examples of
gels used for this are agarose and polyacrylamide.
Gene – the unit of inheritance. Genes are sections of DNA which code for
the production of a particular protein or protein subunit.
Genome – the entire collection of genes in an organism.
DNA Agarose Electrophoresis
Gel
Genotype – the genetic make-up of an organism ie. which versions or alleles of each gene that organism
has.
GFP – Green fluorescent protein – a protein derived from a jellyfish which emits green light when
irradiated with ultraviolet light. GFP is commonly used as a tag to localize substances in molecular
biology.
Glycoprotein – a molecule which contains both protein and carbohydrate components
Golgi Apparatus – a sub-cellular membranous structure involved in the packaging of proteins for
secretion.
Growth Medium – a mixture of nutrients and salts in a buffer liquid which supports
the growth of cells (eukaryotic or bacterial). Growth media may be liquid or a semisolid gel based on agar.
Hormone – a chemical messenger in the body of an organism. Hormones are
generally made in one location and have their effect on cells in another location.
Hormones generally have their effect by binding onto receptors on the outside of
the cell.
Hydrophilic – water soluble.
Hydrophobic –water-repellent.
Hyperproliferation – an abnormal increase in the rate of cell reproduction.
Immunofluorescence – a technique in which a cellular component is localised using an antibody attached
to a fluorescent label.
Kinase – an enzyme which attaches a phosphate group to a protein.
Lesion – any region of abnormal tissue in the body which may be caused by damage or infection.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
67
LB Medium – “Lysogeny Broth” (also “Luria Bertani” after its inventor) – a growth medium used to
culture E. coli in the laboratory.
Ligand – something which binds to another material.
Ligation – a process by which the enzyme DNA Ligase joins together the sugar/phosphate backbones of
two strands of DNA.
Linearised Plasmid – a plasmid that has been restricted so that it is no longer circular.
Lipoplex – a complex formed between a liposome and a nucleic acid – used to introduce genetic material
into cells during transfection.
Liposome – a small “bubble” composed of a phospholipid bilayer folded around to form a sphere.
Liposomes may be used to carry substances into cells by merging with the cell membrane.
Lysate – a “soup” of cellular components made by breaking apart (or “lysing”) cells.
Lysosome – an intracellular body consisting of a membrane surrounding a solution of hydrolytic
enzymes. Lysosomes are involved in the breakdown of foreign particles and some cellular components.
MAPK (“Mitogen Activated Protein Kinase”) – an enzyme involved in the MAPK signal transduction
cascade which promotes the expression of transcription factors which encourage cell growth and
division.
MAPK Scaffold Protein 1 – a protein found in association with caveolae which is involved in ERK
signaling.
Marker – a collection of nucleic acid or protein fragments of known size and composition which are run
alongside test samples during electrophoresis. This creates a series of bands (or a “ladder”) at consistent
locations which can be used to estimate the size of a fragment in a test sample. If a test fragment
migrates the same distance as a fragment of known size, we can say that our test fragment is the same
size as the standard.
MEK (MAPK ERK Kinase) - a protein involved in the MAPK signal transduction pathway. MEK is activated
by the Ras/Raf complex and in turn phosphorylates and activates MAPK.
Motif – a characteristic region of a protein often involved in interactions with other proteins.
MP1 (MEK Partner 1) – a protein found in association with caveolae which is involved in ERK signaling.
Mucosa – a tissue which lines a body surface and which secretes mucus.
Mucus – a substance rich in glycoproteins secreted by cells in the mucosa to provide protection and
lubrication.
Mutation – any change to the normal DNA sequence.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
68
Native – the “normal” or “wild state” version of a gene, as opposed to one that contains a mutation.
Negative Control – a sample put through an experimental protocol as a test of that protocol. A negative
control detects results which look positive in the absence of the variable which could cause a positive
result (i.e. false positives).
Non-polar Amino Acid – an amino acid with a mostly hydrocarbon side chain (ie. few or no oxygen or
nitrogen atoms). Non-polar amino acids are normally hydrophobic.
N-Terminus – the end of a protein chain which has the amino group exposed.
Nucleic Acid - a type of macromolecule consisting of a chain of nucleotides. The sugar and phosphate
groups in the nucleotides form a "backbone", while the nitrogenous bases jut off to the side. DNA and
RNA are nucleic acids. More information on DNA & RNA can be found in Appendix A1 and Appendix A3.
Nucleotide – a chemical group consisting of a phosphate group, a 5-carbon sugar (either deoxyribose or
ribose) and a nitrogenous base (either adenine, guanine, thymine, cytosine of uracil). Nucleotides form
the basis of a strand of DNA.
Nucleus – a membrane bound body inside the cells of eukaryotes which contains the chromosomal DNA.
Oligomer – a molecule consisting of several subunits.
Oligonucleotide – a short sequence of nucleotide bases.
Organelle – a sub-cellular component which carries out a particular task.
Pathology – the study of disease processes.
Pdro – a protein found in association with caveolae which is involved in ERK signaling.
Pellet – the lower (usually solid) phase of a sample after centrifugation.
Peptide – a length of amino acids joined by peptide bonds.
Peptide Bond – the covalent linkage in protein chains between the amino group of one amino acid and
the carboxyl group of the next.
Phenotype – the outward expression of the genes of an organism, as they interact with the environment.
Phosphatase – an enzyme which removes a phosphate group from a compound.
Phosphatidylserine – an important phospholipid found in the cell membrane.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
69
Phospholipid – an amphipathic compound consisting of one or two long, hydrophobic hydrocarbon
“tails” attached to a hydrophilic phosphate containing “head”. Phospholipids are important components
of the cell membrane.
Phosphorylation – the process of attaching a phosphate group to another molecule.
Plasmid – a short, circular sequence of extra-chromosomal DNA. Some bacteria exchange plasmids
between each other, and so plasmids are used as vectors to introduce new DNA into test cells.
Polar Amino Acid – an amino acid with a side chain containing atoms which form polar covalent bonds
(especially oxygen and nitrogen). Polar amino acids are often hydrophilic.
Positive Control - a sample put through an experimental protocol as a test of that protocol. A positive
control detects results which look negative, or a failure of the protocol to give the desired result (i.e.
false negatives).
Prokaryote – an organism whose cells contain no membrane-bound organelles (eg. bacteria, archaea)
Promoter – a region of DNA which encourages the transcription of a subsequent gene.
Protease – an enzyme which breaks down a protein.
Protein – a large biological molecule composed of a chain of smaller molecules called amino acids.
Proteins perform a range of roles in the cell, including structure, catalysis of chemical reactions,
recognition of substances and regulation of cellular processes. More information on proteins and protein
structure can be found in the General Information section.
Protocol – a series of standard experimental procedures followed in the laboratory.
Punctate – having a “spotty” appearance.
Raf (“Rapidly accerlerating fibrosarcoma”) – a protein involved in the MAPK signal transduction pathway.
Raf binds to and is phosphorylated by Ras and in turn phosphorylates and activates MEK.
Ras (derived from “Rat sarcoma) – a protein involved in the MAPK signal transduction pathway. Ras is
activated by the binding of adaptor proteins to tyrosine kinase receptors and it binds to and activates
Raf. The complex then phosphorylates MEK.
Receptor – a protein molecule embedded in the cell membrane which binds to a substance outside the
cell. When this substance is bound, a change in the receptor protein triggers changes in other proteins
within the cell which then influences metabolic and regulatory processes inside the cell.
Repressor – a region of DNA which inhibits the transcription of another gene.
Restriction Digest – a technique in which an enzyme is used to cut the DNA at specific points. Restriction
enzymes bind to specific sequences of nucleotides in the DNA and then cut the sugar-phosphate
backbone of the molecule.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
70
Selective medium – a type of growth medium which only allows a certain organism to grow. For
example, a culture medium may contain a specific antibiotic (e.g. Ampicillin). The only organisms which
can grow in that medium are those which possess the gene for resistance to that antibiotic. This means
that common contaminating organisms may not overwhelm the growth of the organisms we are trying
to grow. In molecular biology, we often include an antibiotic resistance gene along with the gene we are
trying to express. This means that only organisms which contain the resistance gene (along with the gene
of interest) can grow, and therefore we can subculture from these populations confident that all of the
organisms have been transformed.
Signal Transduction – the process of transferring a signal from outside the cell to inside the cell using
specialised proteins.
Signal Transduction Cascades – the metabolic pathways through which an extracellular signal causes a
response within the cell.
Spectrophotometry – a process in which light of known intensity and wavelength is passed through a
solution and used to estimate the levels of substances dissolved in that solution. Different materials
absorb light of specific wavelength and the more of that substance present, the more light of that
wavelength will be absorbed. If we measure the amount of light of a particular wavelength which passes
through the substance, we can calculate the amount absorbed and therefore estimate the levels of the
material absorbing the light.
Sphingolipid – a family of lipids found in the cell membrane which play a role in signal transduction and
cell recognition.
Squamous Cell Carcinoma (SCC) – a cancer which affects squamous epithelium, a tissue which consists of
layers of flattened cells (eg. the skin, the lining of the mouth).
Steroid – a group of chemical compounds with a common structure based on a series of four interlocking
carbon ring structures (called a sterane). Steroids differ from each other by the functional groups
attached to this central structure. Examples of steroids include cholesterol and the hormones
testosterone and oestradiol.
Subunit – in a protein with quaternary structure, a subunit is one of the individual protein chains which
make up the larger structure.
Supercompetent Cells – bacteria capable of taking in DNA from their environment with high efficiency.
Commercially, supercompetent cells are usually defined as those capable of >109 transformations per μg
of transforming DNA.
Supernatant – the upper (usually liquid) phase of a sample following centrifugation.
Testosterone – the principal male sex hormone. Testosterone is an example of a steroid.
Tissue – a collection of cells working together to carry out a particular function in the body.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
71
Transcription – the process of creating an mRNA molecule from a DNA template.
Transcription Factor – an agent which promotes the transcription of genes which code for other
proteins.
Transformation – the transfer of genetic material into a bacterial cell.
Translation – the process of creating a protein following information encoded in a mRNA molecule.
Tyrosine Kinase Receptors – a family of proteins embedded in the cell membrane which are involved in
signal transduction. When a ligand binds to their extra-cellular domains, they attach phosphate groups to
proteins inside the cell which then trigger signal transduction cascades.
Tumour – a mass of tissue, often consisting of abnormal or undifferentiated cells. Tumours may be
considered as benign or malignant.
Ultraviolet Light – electromagnetic radiation with a wavelength between 400nm and 10nm.
Upstream – sequences of amino acids in proteins are always written from the N-terminus to the Cterminus. If a sequence is said to be inserted “upstream” from a target gene, this means that it is
attached before the N-terminus.
Vector – something used to transfer DNA into a target cell.
Vesicle – a sub-cellular membrane-bound body.
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
72
Notes :
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
73
Notes :
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
74
Notes :
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
75
Notes :
© 2010 State of Queensland (Department of Education & Training)
SPARQ-ed – The University of Queensland Diamantina Institute
Princess Alexandra Hospital, Woolloongabba, Australia.
http://www.di.uq.edu.au/SPARQ-ed
ph. +61 7 3176 7868 fax. +61 7 3176 5946
Email: [email protected]
76