The Coca – Cola Company Market Position Analysis 1 The Coca – Cola Company Market Position Analysis 2 Market Position Analysis Dwayne Woods Capstone Experience in Integration & Strategy Dr. Thomas H. Kemp December 18, 2013 Assignment 3: Market Position Analysis: The Coca-Cola Company The Coca-Cola Company is a leading world brand that makes soft drinks that are ready to drink. The company makes products such as Coke, Fanta, Sprite, Minute Maid, Dasani water among others. It has managed to hold its market position as the leading beverage producer in the world, facing competition from PepsiCo. Coca-Cola sells its products in the whole world, having divided its operation areas in regions such as Africa, Eurasia, Latin America, European Union, North America and Pacific. The company’s mission statement is ‘refreshing the world –in mind, body and spirit’. Coca-Cola Company’s target market is any person who likes soft drinks, but lately they have been targeting teenagers and people below thirty. In short, its main target market is the youth. According to Vendredi (2012), the company uses several strategies to reach its target market. The company has partnered with restaurants and fast foods such as McDonald. This is because most of these young people eat in such places. The company has also made adverts for their current slogan, ‘Open happiness’ with teenagers as the cast. Most of the adverts show young people below the age of thirty having fun while taking their products. Their target market consists of both male and female youth. The young people are seen buying lots of beverages and Coca-Cola has set out to have them as their target audience. Coca-Cola does not segment its market by age. From, an interview with a Coca-Cola manager, she claimed that their market is undifferentiated. Ali and Mohammad (2011) argue that their market is segmented on the basis of geographic region, demography, climate and behavior. Geographically, it sells higher quality products in the developed nations since per capita income is high. It also sells more in urban areas around the world than in the rural areas. The company segments its market according to climate because it sells its products in hot areas more than in cold areas. All its products are served cold. During summer, Coca-Cola sales are high in most areas around the world. The company also segments its market according to demographics. It has products for children between 4 and 12 since it has some that have flavors such as cherry, vanilla and lime. The Coke, Fanta and Sprite brands mostly targets the youth. The company also packs its products for families, that is, in the economy pack that usually has six cans. It also segments by income and this is seen in packing for example, plastic bottle soda is more expensive that the glass bottle. The Coca-Cola Company is positive that their products satisfy their customer’s needs. This is because their customers are loyal. They also know this through ...

1. The Coca – Cola Company
Market Position Analysis
1
The Coca – Cola Company
Market Position Analysis
2
Market Position Analysis
Dwayne Woods
Capstone Experience in Integration & Strategy
Dr. Thomas H. Kemp
December 18, 2013
Assignment 3: Market Position Analysis: The Coca-Cola
Company
The Coca-Cola Company is a leading world brand that makes
soft drinks that are ready to drink. The company makes products
such as Coke, Fanta, Sprite, Minute Maid, Dasani water among
others. It has managed to hold its market position as the leading
beverage producer in the world, facing competition from
PepsiCo. Coca-Cola sells its products in the whole world,
having divided its operation areas in regions such as Africa,
Eurasia, Latin America, European Union, North America and
Pacific. The company’s mission statement is ‘refreshing the
world –in mind, body and spirit’.
Coca-Cola Company’s target market is any person who likes
soft drinks, but lately they have been targeting teenagers and
people below thirty. In short, its main target market is the
youth. According to Vendredi (2012), the company uses several
strategies to reach its target market. The company has partnered

2. with restaurants and fast foods such as McDonald. This is
because most of these young people eat in such places. The
company has also made adverts for their current slogan, ‘Open
happiness’ with teenagers as the cast. Most of the adverts show
young people below the age of thirty having fun while taking
their products. Their target market consists of both male and
female youth. The young people are seen buying lots of
beverages and Coca-Cola has set out to have them as their target
audience.
Coca-Cola does not segment its market by age. From, an
interview with a Coca-Cola manager, she claimed that their
market is undifferentiated. Ali and Mohammad (2011) argue
that their market is segmented on the basis of geographic
region, demography, climate and behavior. Geographically, it
sells higher quality products in the developed nations since per
capita income is high. It also sells more in urban areas around
the world than in the rural areas. The company segments its
market according to climate because it sells its products in hot
areas more than in cold areas. All its products are served cold.
During summer, Coca-Cola sales are high in most areas around
the world. The company also segments its market according to
demographics. It has products for children between 4 and 12
since it has some that have flavors such as cherry, vanilla and
lime. The Coke, Fanta and Sprite brands mostly targets the
youth. The company also packs its products for families, that is,
in the economy pack that usually has six cans. It also segments
by income and this is seen in packing for example, plastic bottle
soda is more expensive that the glass bottle.
The Coca-Cola Company is positive that their products satisfy
their customer’s needs. This is because their customers are
loyal. They also know this through the interaction with their
customers on social media. The company’s website also has a
segment left for customers to post their complaints and
comments on their products. The company takes care of all their
customers’ needs since they have different flavors to suit their
consumers’ tastes. For those who don’t take soda, the company

3. makes fruit juices by the brand name of Minute Maid. The
company has however been unable to provide low calorie and
low sugar soda in all their flavors to the customers. Only the
Coke brand has a low sugar and calorie product know as Coke
Zero.
Coca-Cola’s main competitor is PepsiCo. PepsiCo makes drinks
such as Mirinda, Pepsi, 7Up, Gatorades and Tropicana fruit
juice among others. From these two companies, their most
famous brands are the cola drinks, with Coca-Cola’s going by
the brand name Coke while that of PepsiCo is Pepsi. Let’s
compare these two drinks. In the USA, their price is the same
with the 2 liters bottle going for $1.79. The two brands have
several products produced under them. For coke there’s Diet
Coke, Coke Zero and Coke Classic. The Pepsi portfolio has
Pepsi, Diet Pepsi and Pepsi-MAX. According to Lubin (2012)
Pepsi was found sweeter in a sip test. He argues that Pepsi has a
citrus flavor burst while Coke has a vanilla-raisin taste. Both
are made with almost the same products but Coke is more
carbonated than Pepsi, Pepsi has more caffeine, sugar and
calories while coke has more sodium. Their qualities are
difficult to differentiate since they taste almost the same, but
each brand has its loyal customers. They are both in the market
to quench the thirst of their customers. Coke is found world-
wide while Pepsi is not available in all countries. Coke is easily
available even in remote areas but in some countries Pepsi is
only available in leading supermarkets. Coca-Cola is branded in
white and red while Pepsi has blue as the major with red and
white. Coca-Cola is more valuable than Pepsi with the former
worth $79 billion while Pepsi is worth $17 million (Anuar
2013). Coca-Cola is associated with fun and happiness. Pepsi
has branded itself using celebrities such as Beyoncé, Britney
Spears and Michael Jackson and good music.
Coca-Cola’s competitive advantage lies in their image and
availability. Their products are available worldwide and are
affordable in every country since pricing is different in each
country. The image of the company as well as that of their

4. products has given it a first place before Pepsi. The company is
known to have good products and to be a world leader in
beverage production. Their products are advertised in fun-filled
adverts that target its market segment. They also are packed in
well-designed packs that are attractive to the consumer.
The Coca-Cola Company has a sustainable competitive
advantage. Its competitive advantage stems from the Coca-Cola
brand itself. It is a known brand in the whole world. Its other
sources of competitive advantage are; the secret recipe,
developing new products and re-inventing new ones and its
production techniques that have produced high quality products
at low costs (So Opinionated 2012). The company is able to
sustain its competitive advantage through re-branding and re-
inventing its products and associating them with fun things.
The company has therefore been able to hold a top position
against its competitors in the whole world. Even during the US
economic boom, Coca-Cola made negligible losses since it sold
in other continents and its sales in the US changed slightly. It is
also a highly valuable company worth billions of dollars.
Table Showing Coca-Cola’s Market Position
Coca-Cola
Pepsi
Satisfaction
2
1
Refreshment
2
2
Health
1
0
Quality
2

5. 2
Sweetness
1
2
Total
8
7
References
Ali, S. and Mohammad, S. (2011). Target market and market
segmentation of Coca-Cola. Retrieved from:
http://www.slideshare.net/sikander22/target-market-and-market-
segmentation-of-cocacola
Anuar, D. (2013). Comparison of brands: Pepsi Cola vs. Coca
Cola. Retrieved from:
http://www.academia.edu/4977803/Comparison_of_brands_Peps
i_Cola_vs_Coca_Cola
Lubin, G. (2012). Here’s the real difference between Coke and
Pepsi. Retrieved from: http://www.businessinsider.com/the-
difference-between-coke-and-pepsi-2012-12
So Opinionated. (2012). The Coca-Cola business model and
their competitive advantage. Retrieved from:
http://sopinion8ed.wordpress.com/2012/11/23/the-coca-cola-
business-model-and-their-competitive-advantage/
Vendredi. (2012). Coca Cola targeting and positioning.
retrieved from:
http://softdrinkcolawar.blogspot.com/2012/12/coca-cola-
targeting-and-positioning.html
Ed aDear writer:
This work is about a practical report which is based on these
series of procedures that were performed on different days. For

6. example practical one was performed on day 1, prac 2 was
performed on day 2 and so on.
The guidance is on the very last page.
I was group six in this experiment. I have included the results
for each part of the practical after the protocol for each section(
some results are provided separately, because they were too
large to include in this document. E.g. microarray results). And
as you can see there are other groups present as well. In result
analysis, you should analyse my results, compare it to what is
normally expected(control), and briefly discuss other groups’
results.
I have made notes during the practical and have highlighted
them in red so it can be distinguished.
In each practical regents/substances were used and in the
introduction you need to mention why these were used and what
are their roles. In some practicals, I have highlighted what
further explanations are needed for that particular practical.
This part needs to be 5000 words long and the instructions are
provided at the very last page. You are required to provide
enough and adequate referencing.
Practical for Microarray technology
Aims:
The aim of this practical is to provide an understanding of the
modern biotechnology in functional genomics and cancer
biology. These approaches are revolutionizing the biomedicine
technology, especially in cancer research, influencing diverse
areas of biological research from vaccine through to drug
discovery process. The practical will provide the opportunity
for the students to explore the key technology for modern
molecular biologists - Microarray technology, which has been
widely used in different biomedical field including cancer
research; To be familiar with the techniques used in functional

7. genomics analysis and how large-scale gene functional analysis
programmers are designed and implemented in genomic
research; To increase the competences required in modern
laboratory skills.
Objective
To study the gene expression profile using Microarray
technology.
--- Specifically, you (working in pairs) will compare T
lymphocytes (mouse MF2 or human Jurkat cells) with the same
cells but transfected with a gene called Egr-2 (eg, Egr2/MF2,
MF2 cells with a Egr2 gene) to see whether there is any
difference of gene expression profile.
Practical arrangementWeek-1 Practical
Week-2 Practical
Date
Practical topics (13:00-17:00)
Lab135
Practical topics (13:00-17:00)
Lab135
Monday
Tuesday

8. Wed.
Microarray practical-1
· RNA extraction
· Measuring RNA concentration
Microarray practical-2
· Making cDNA and Labeling
· RNA on agrose gel
Microarray practical -3
· cDNA purification
· Quality control check
Microarray practical -4
- Array chip pre-hybridization
- Array chip hybridization
Microarray practical -5
- Array chip washing
- Array chip scanning
Microarray practical -6
-Array analysis: bioinformatics

9. - Instruction for Practical report
Practical Report:
You need to hand in your practical report (80%) together with
your log book (20%)
Microarray practical-1:
Samples:
- Each pair is provided with one of the following test sample
(mouse MF2-T cells transfected with Egr2 gene) for RNA
extraction.
Note: RNA will be degraded if not properly handle it!
-Always wear gloves throughout the procedure and avoid
making contact with anything (ensure that everything is
organised for the experiment beforehand).
- Place paper towel on bench working area before starting the
experiment
-Avoid contact of pipette tips with anything when carrying out
the experiment
Procedure-1: RNA extraction with TRI reagent
1. The cells are already with 500 ul TRI reagent (lysis
buffer)(dissolves the cells, so that all the RNA/all componants
can be available) on ice
2. Allow the sample to stand at room temperature for 5
minutes.
3. Add 100 ul of chloroform (200ul per 1 ml of TRI reagent
used)(to separate RNA,DNA from other molecules) to each

10. sample followed by vortexing to mix the sample for 15 seconds.
4. Leave the sample to stand at room temperature for 15
minutes.
5. Centrifuge(to separate different materials based on their
molecular weights) the mixtures (12000 RPM, 4 Degrees C for
15 minutes).
NOTE: It should be expected for each of the samples to form 3
phases upon centrifugation; a red organic phase containing
protein (bottom-most layer), an inter-phase containing DNA
(middle layer) and a colourless aqueous phase containing RNA
(upper-most layer).
6. Transfer the upper-most aqueous phases from sample to
fresh eppendorf tubes and then add 250 ul of isopropanol(to
precipitate and in solid form) (500ul per 1 ml of TRI reagent
used) to the sample.
7. Mix the sample and then allow to stand at room temperature
for 10 minutes.
8. Centrifuge the sample (12000 RPM, 4 Degrees C for 10
minutes).----make a mark on the one side of tube, so you know
where you expect to see the RNA pellet! because following
centrifugation, the RNA precipitate will form a pellet on the
side and bottom of the tube for the sample.
9. After centrifuging, remove the supernatant and then the RNA
pellet should be washed by adding 500 ulof 75% ethanol. (
washing solution, to get rid of excess)
10. Mix the sample thoroughly by vortexing and then centrifuge
(7500 RPM, 4 Degrees C for 5 minutes).(repurify)
11. Remove supernatant from sample as much as possible
(otherwise it will not be dissolved in the next step!)

11. 12. Air-dried for 10 minutes.( to remove all ethanol by adding
water, the RNA will be dissolved)
NOTE: When drying the RNA pellets, care should be taken to
ensure that the pellets do not dry completely, as this would
decrease the solubility of the pellets !
13. Finally, add 100 ul of DEPC (Di-Ethyl-Pyro-Carbonate)( to
dissolve RNA in water). treated water to sample pellet. To
facilitate dissolution, mix sample by repeated pipetting for 5
minutes.
Note: You need to take 5ul from the RNA sample for running
agarose gel (at procedure-4, Practical -2 tomorrow), label the
tube with your group number andgive it to PHD Student(staff)
.the results are shown below: the first picture is a high
resolution picture and the second picture is a low resolution .
Note: 5ul from all the samples were loaded. The order of the
samples from
Left to the right are following:
Control, 2, 3, 4, 5, 6, 7, 8, VR9, 10, 11, 13, 14, 16, YS, ?(no
labelling) , ladder.
(it seems no tubes labelled with 1, 12, 15). Please note that we
are group six
Only RNA shows distinctive bands ( typically two bands)
The above band is S28
The below band is S18
Control shows strong bands and the S28 must be brighter. If
there is extra band liquidation has happened.

12. 5ul from the RNA sample where taken to spectrometry, the
results are shown below. There was a problem with this stage,
as a few groups that had strong bands in the agrose gel in
contrast to us who had were weak bands didn’t get a good ratio
but our group did. I think it was either the machine or the
staff(PHD student)
The above result is for my group which was group six. The
calculations and procedure is further explained below:
Procedure-2: Measuring RNA with spectrophotometer
-Use a spectrophotometer in order to determine the quality of
the extracted RNA samples. It is noteworthy that the quality of
RNA is one of several important determinants, governing the
success of micro-array experiments.
- The criteria for determining the purity of RNA is based on the
260/280 ratio which is a measure of the extent to which the
RNA sample is free from DNA and protein. A 260/280 ratio of
> 1.7 indicates that the RNA is of acceptable purity/quality.
- Procedure to measure the purity of each of the extracted RNA
samples,
1) 990 ul of water add in a corvette.
2) 10 ul taken from your RNA sample and add to the same
corvette, mix well.
3) The cuvette will be then placed in the complementary slot on
the spectrophotometer and record the following from the
spectrophotometer:
a) Record the reading for the ration: 260/280.
b) Record the reading for the RNA concentration in the corvette
(ng /ul)
4) Calculate the actual concentration of your RNA sample (ug
/ul).

13. - The following table shows the results of the calculations as
some examples with dilution factor: 100 (10 ul in 1000 ul of
water: 1000/10=100)
Reading (examples)
Concentration ( ug/ ul)
260/280 Ratio (examples)
11.2 ng/ul
11.2 X 100 (dilution factor) / 1000 = 1.12
2.17
6.8 ng/ul
6.8 X 100(dilution factor) / 1000= 0.68
1.96
Note:
1) Y need to have total RNA for one sample about 50 ug for the
experiment,
so if the total RNA (total ul x concentration ug/ul) is < 50 ug,
then you need to get relevant RNA from Dr. Li.
2) You need to dry down the sample if the RNA concentration is
< 0.5 ug/ ul, because you will need about 40-50 ug in a volume
less than 8 ul, which will be required for the Practical-2
(tomorrow)
You need to talk about other groups’ results as well as group
six. And that why results from spectrometer and gel reflect
different outcomes
.
Procedure-3: To get more concentrated RNA (it was done by
the DR.li and provided to groups who did not have good RNAs
including our group this was because our concentration was
21.76 and it was less than 40 ug)
1) Take about 40 ug (40-50ug) ug of your RNA in one
eppendorf tube-1 (how much volume you need? e.g if your
sample contraction 1.12 ug/ul, then you will need the volume:
40 /1,12= about 40ul),

14. 2) Take another eppendorf tube-2 with water as control to
measure the volume for the evaporation, which should be the
same volume as tube-1,
3) Label with your group number
4) Put both tubes (water tube and sample tube) with opened lid
in a rotator evaporator (located at Lab 128),
5) make a mark on the one side of tube, so you know where you
expect to see the RNA sample!
6) The rate for the evaporation is about 1ul/min. About every10
min, check the volume for the water tube (tube-2) until the
volume is 8 ul or less.
Microarray practical-2
Procedure- 4: Cyscribe First-strand cDNA Labelling
- You should have two tubes: one containing control RNA
sample: (MF2 RNA or Jurkat RNA) which is on the ice already
and the other containing test RNA sample (you extracted) for
this experiment. Both need the volume </ or = 8ul of
RNA/sample.
The gene we are looking for is Egr2. T-cells without the gene
and T-cells with the gene. The extracted RNA is to be converted
to cDNA because cDNA is more stable.
- THIS MUST BE DONE FOR EACH OF THE 2 TUBES!!!!
A) Primer annealing for each of two RNA:
1.
8 ul RNA (add water if less than 8ul)
+
3 ul Oligo-DT( primer, initiative)
----------------------------

15. 11 ul in total
2. Mix gently by pipetting up and down.
3. Incubate reactions at 70 degrees C for 5 minutes (use heating
block).
4. Cool reactions at room temperature for 10 minutes to allow
the primer and the mRNA template to anneal.
5. Spin down reactions for 15 seconds in a micro-centrifuge.
B) Extension Reaction:
1) Add the total 9ul of “cy-3 mix” to the 11 ul of control
RNA(MF2 RNA or Jurkat RNA)
2) Add the total 9ul of “cy-5 mix” to the your extracted 11 ul of
test RNA
Each mix contain:
5 X Cyscribe Buffer
4 ul
0.1 M DTT
2 ul
dCTP Nucleotide Mix

16. 1 ul
dCTP Cye-Dye-Labelled Nucleotide
1 ul
Cyscribe RT (enzyme)
1 ul
The only difference for the mix are:
Cy-3-dCTP used for the control RNA, red fluorescence
Cy-5-dCTP for the test RNA. Green fluorescence
Total = 9 ul (above reagents) + 11 ul (RNA + Oligo-DT) = 20
ul.
3. Mix by gentle pipette, then centrifuge for 30 seconds in
micro-centrifuge.
4. Incubate the reactions at 42 degrees C for 1.5 hours. (after
incubation, place reactions on ice for next step)
Note: during the incubation, you need to do procedure-5
Procedure- 5: RNA quality check by argrose gel-
demonstration
- You will see the demonstration how to run the agrose gel when
you are doing procedure-3B.
- In the end of the experiment today, you will get the picture for
your RNA taken from the agrose gel and you need to analysis
the RNA quality to see how much degradation of the RNA.
Procedure-6: Degradation of unlabelled RNA

17. (continue from procedure-4):
1. Adjust water bath to 37 degrees C.
2. Add 2 ul of 2.5 M NaOH to each cDNA reaction from above
procedure-3B.
3. Mix by vortexing and spin for 15 seconds in a micro-
centrifuge.
4. Incubate reactions at 37 degrees C for 15 minutes
5. Add 10 ul of 2 M HEPES-free acid to the reactions
(neutralises NaOH)
6. Mix by vortexing and spin for 15 seconds in micro-centrifuge
7. The reactions are now ready for purification (practical -3,
tomorrow), give to Helen to store in -20.
Microarray practical -3
Procedure- 7: Purification of Labelled cDNA:
Purification was perfomed to get rid of extras such as liquidated
RNA, enzyme, nuecluetides, fluoerescnece molecules and etc
1. Add 500 ul of capture buffer(tries to capture cDNA, makes
cDNA precipitate) to 1 GFX column (allows small molecules to
past through and axts like a membrane)
2. Add both cDNA samples (already with Cye-5 and Cye-3 in
them) into the column and mix
3. Centrifuge the column – 13,000 rpm – 30 seconds
4. Add 500 ul of wash buffer(ethanol, as it cannot be water as
RNA is dissolved in water) to the column – centrifuge 13,000
rpm, 30 seconds

18. 5. Discard liquid in collection tube
6. Add 500 ul of wash buffer to the column – centrifuge at
13,000 rpm, 30 seconds
7. Discard liquid
8. Place column back into the collection tube + centrifuge at
13,000 rpm for 30 seconds
9. Transfer column to a fresh eppendorf tube + add 30 ul of
elution buffer (water to dissolve) to the tip of the column
10. Incubate at room temperature for 5 minutes
11. Centrifuge at 13,000 rpm for 1 minute
12. You will get about 30ul of elutes after centrifugation. Take
1ul of elutes for labeling efficiency check at procedure-8.
12. Dry down the cDNA using a rotator evaporator until less
than 1 ul. (the procedure is the same as in procedure-3, make a
mark on the one side of tube, so you know where you expect to
see the pellet!!!
Procedure- 8: Labeling efficiency check with Nonodrop
instrument (performed for us by laboratory staff)
· You will bring your 1ul of elutes to room 120 (the same floor,
but inside Cancer Research Institute)
(note: Use the provided lens tissue to clean the nanodrop. DO
NOT USE OTHER TISSUE TYPE AS THIS WILL DAMAGE
THE LENS, please dispose of the tips and tissues into the
designated tips box and biological waste bag respectively)
PRECEDURE FOR MICROARRAY DYE QUANTIFICATION
1) Select Nanodrop 2000 from the desktop icon
2) Choose microarray as the type of measurement required

19. 3) Click OK in the wavelength verification dialogue box
4) Clean the nanodrop lens and then adding a 1ul of ddH20,
blank the nanodrop
5) After Blank, the MEASURE icon will go green, enter sample
ID e.g. group 1, select ssDNA, leave add report clicked, load
1ul of sample and measure. Remember to change sample ID
between readings
6) After readings have been taken, to export files go to report→
ctrl A (to highlight all files) → save in pre-determined location
as a report, excel XML file. To view, Open files normally as
you would an excel document
7) To save graphs, press ‘Print screen’ on key board on page
with graph, export to a PowerPoint document and CROP the
image
Microarray practical -4:
Note:
8) Each group will be provided an mouse array chip or human
array chip which contains mouse or human genes. Each gene
was arranged in a specific position on the glass slide, and you
will use this chip to hybridizated with your cDNA
9) Avoid to touch the surface of the chip which contains the
DNA genes.
10) Health and safety warning about FORMAMIDE contained in
prehybridization or hybridization buffer. Which is HARMFUL
IF SWALLOWED, INHALED OR ABSORBED THROUGH
SKIN. CAUSES IRRITATION TO SKIN, EYES AND
RESPIRATORY TRACT. AFFECTS CENTRAL NERVOUS
SYSTEM. MAY AFFECT THE REPRODUCTIVE SYSTEM.
Procedure- 9: Pre-hybridization of chip (to get rid of
background)

20. 1. Set water bath at 42 degrees C
2. label the chip with test sample information (eg. Egr-MF2 or
Egr-Jurkat) and group number, and the date.
3. Add 500 ul of pre-hybridization buffer to middle of the chip
(be cautious of air bubbles)( to reduce background)
4. Place the cover-slip carefully on the chip (make sure
coverslip is over the chip part of the slide). Place cover-slip
horizontally on chip so that hybridization buffer covers the chip
and air bubbles are expelled out
5. Give the chip to Helen/Moshin, and the chip will be put into
cassette and incubate at 42 degrees C for 1-2 hours until you
prepare the stuff for hybridization step see below. Make sure
the cassette is sealed!
Procedure- 10: Hybridization of the chip (cDNA hybridizated
with DNA genes):
1. Re-suspend cDNA (find the pellet first!) with “blocking
mix” which contains 6 ul of Cot DNA+1.5 ul of oligo-dA
2. Denature at 95 degrees for 2 minutes (use heat block)
3. briefly centrifuge 10 seconds
4. incubate at 75 degrees C for 45 minutes (use heat block)
5. Add “hyb-mix”(which contains 7.5 ul of hybridization buffer
+ 15 ul of 100% formamide) to the above tube. Mix well + spin
for 15 seconds
6. Take off cover-slip (do not touch chip) from the pre-
hybdizated chip (get from Helen/Moshin)
7. Add whole sample from step-5 to the middle of the chip.
Place cover-slip carefully on the chip, then give to Anne
8. Incubate the chip in a chamber , overnight in 42 degrees C
water bath.
THE COVER BROKE AND WAS DROPPED!!
Note:

21. Please bring thememory stick /or one CD tomorrow, so we can
save the image for you when we scan the chip for your results.
Microarray practical -5
Procedure- 11: Washing (to get rid of unbound cDNA) and
Scanning
1. Add about 300-400ml of 2X SSC (SALINE SODIUM
CHALE) buffer into the wash container, (buffer: balance low,
easy to wash away)
2. Place chip in the wash container slot, let cover-slip go away
it, and
Use the R100 – Luckham shaker to perform the following
washes (Washes to be accurately timed. DO NOT LEAVE IN
WATER AT THE END.
The slides were diped into these solutions:
Buffer (300-400ml)
Time
No of times to be dipped into
1 X SSC + 0.2% SDS( SODIUM DOCTECYL SALFALE)
5 minutes
X 1
0.1 X SSC + 0.1% SDS
0.1 X SSC
5 minutes
5 minutes
X 1
X 1
3. After final wash, empty wash container + replace with 2X
SSC. Place the chip into it
4. Place chip in 50 ml tube, put it in the centrifuge, make sure it
has been balanced !

22. 5. centrifuge 1000 rpm for 20 seconds, Cover tube with
aluminium foil
7. The chip is now ready for scanning.
Note:
1) Please bring the memory stick /or one CD with you, so we
can save the image for your results!
2) We will do the scanning in the Microarray Lab in Cancer
Institute.
Microarray Practical – 6 a separate powerpoint presentation is
provided. This presentation was presented to us by PHD student
and explained how to do this. We performed this. I have
included the files that this procedure provided us with. You
need to mention the use of this procedure which then comes
useful in practical report. (stage 14)
Procedure – 12: Array analysis- Using GoMiner to categorise
genes according to their biological functions
Purpose of Software:
Typical microarray experiments generate vast amounts of data,
making it difficult to interpret for the biologist. If the biologist
wishes to extract biological meaning from such overwhelming
data, the data itself must be further mined. GoMiner is one of
many tools used to “make sense of the data”. More specifically,
it categorises genes from any given data set into their respective
function at the biological, cellular and molecular levels.
Accessing GoMiner:
To access GoMiner, take the following steps:
1. Open “Internet Explorer”
2. Enter the following http address: -
http://discover.nci.nih.gov/gominer/GoCommandWebInterface.j

23. sp
Using GoMiner:
When GoMiner is opened, the user will be prompt with the
following options:
Step 1:
“Select total file”
· To load the gene-file of interest, click on Browse
· For the purpose of this practical, use the “down reg T2 genes
for gominer.txt” file.
· To select the files go to “L:drive (depapps on ‘academic.
Windsor’)
double click on “BL” folder
then double click on “GoMiner” folder
Select the gene of interest file by double clicking it.
Step 2:
“Select changed”
· For the purpose of this practical select the same file as the
Total file, following the above instructions.
Step 3:
“Select DataSource(s)”
· Make sure “Choose data source from List” is selected.
· “All” must be highlighted in the selection box.
Step 4:
“Select organism(s)”

24. · Select “M. musculus”
Step 5:
“Select Evidence Code(s)”
· Select “All”
Step 6:
“Select Lookup Setting”
· Both “Cross Reference” and “Synonym” boxes should be
ticked.
Step 7 to 10: leave all the settings for these steps as they are..
Step 11: “Select Root category for evaluating enrichment
ratios, Fisher’s Exact and the FDR’s”
· Select “All/Gene ontology”
Go to Step 13:
“Your E-Mail Address”
· Enter your Brunel email address.
Step 14: “Submit Query”
· Click on Submit.
· It will take 5 to 20 Minutes to receive the results by email.
REPEAT THE PROCEDURE FOR THE UP-REGULATED
GENES.
Once you have received the email from [email protected] . Click
on the hyperlink that lets you browse your results (see below).

25. The following page will open on your screen. Click on “Archive
Results (Zip)” file link. Shown below.
A prompt will appear Click on “Open”.
The files will open in PowerArchiver 2001. To obtain you file
click on the following excel file:
“CORRECTEDNAME_down-
reg_T2_genes_for_gominer.txt.change.gce.xls” file or
“CORRECTEDNAME_up-
reg_T2_genes_for_gominer.txt.change.gce.xls” file.
For the purpose of this practical you are only interested in
Columns A and B. You can delete columns C to J.
Save this file onto your USB stick.
Instruction for practical report
The main sections of the report should be as follows:
1. Title
2. Abstract
3. Introduction to include definition of microarray, its
application, principle, pros and cons
The biological question is gene profile from Egr-2
4. Material and Method
5. Results - You need to include at least four results including
the pictures/or table produced during the experiment:
a) RNA quality and quantity

26. b) cDNA labelling efficiency check
c) Array chip hybridization results
d) Present the results produced from the Gominer software
analysis.
6. Discussion
13. You need to discuss your result produced from the practical
14. Also select any 5-10 genes from by Gominer analysis, which
are relevant to your experiment system, and search on the
internet for the information to explain why they are highly
expressed or lower expressed or no changes.
15. Discuss why this technology and some other technologies
such as transgenic technology, chip sequence can be used for
cancer research or drug discovery.
7. References
8. Appendices
Note:
the practical report should be in great scientific dept and shows
extreme knowledge of the field for very good mark.
Please do not hesitate to contact me if you are unsure of any
thing.. In practical report, the procedures should be mentioned
but as the word limit is 5000 , try to compact the methodology
without losing marks.
Click on this hyperlink.
Click on this hyperlink.
Click on this hyperlink.

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