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Unit 18 - Assignment 4 - P6
DNA Extraction
Equipment:
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Sodium chloride solution 1cm3 of 3% (3gm in 100cm3 water)
Detergent 1 cm3 – cheap, watery variety - not concentrated
5cm3 (drinking) water in plastic cup
Ice cold ethanol
Test tubes & racks
Dropping pipettes
Containers of hypochlorite bleach or ‘Virkon’ for disposal of tubes

Step by step:
1
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3
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5
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Gently chew the inside of the mouth for 30 secs
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Swill 5cm3 of water around your mouth vigourously for 30 seconds
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Do not shake hard!
6
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Slowly pour 5cm3 of ice cold ethanol down the side of tube
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Place tube in rack, and observe
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Slowly, small strands of DNA should appear at the interface of the ethanol and the lower
mixture, then rise up through the ethanol
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Electrophoresis
Method:

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Dye C travelled the furthest in the gel
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The type of DNA that each dye represents:
Dye A = Total bacterial DNA
Dye B = Plasmid DNA
Dye C = Gene fragment DNA
Dye D = Ligated plasmid DNA

Cell transformation
Method:
1
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Label both tubes with your group’s
name
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2
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3
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Do not use cubed ice
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Use a sterile loop to pick up a single colony of bacteria from your starter plate
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Spin the

loop between your index finger and thumb until the entire colony is dispersed in the
transformation solution (with no floating chunks)
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Using a new sterile loop, repeat for the -pGLO tube
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Withdraw a loopful
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This is similar to
seeing a soapy film across a ring for blowing soap bubbles
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Optionally, pipet 10 µl of pGLO plasmid into the +pGLO tube and
plasmid DNA -pGLO mix
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Do not add plasmid
DNA to the -pGLO tube
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5
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Make sure to push the tubes all the way down in the rack
so the bottom of the tubes stick out and make contact with the ice
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While the tubes are sitting once, label your four agar plates on the bottom (not the lid) as shown
on the diagram
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Heat shock
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Make sure to push the tubes all the way down
in the rack so the bottom of the tubes stick out and make contact with the warm water
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For the best transformation results,
the change from the ice (0°C) to 42°C and then back to the ice must be rapid
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8
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Remove the rack containing the tubes from the ice and place on the bench top
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Repeat
with a new sterile pipet for the other tube
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9
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Using a new sterile pipet for each tube,
pipet 100 µl from each of the tubes to the corresponding plates, as shown on the diagram onto
the appropriate plates
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Use a new sterile loop for each plate
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11
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Put your group name and class period on the
bottom of the stack and place the stack upside down in the 37°C incubator until the next day
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live
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aspx?cid=109322502356c806&page=view&resid=109322502356C80
6!2068&parId=109322502356C806!1574&app=Word

Prediction of bacteria
pGLO
+
NB – No bacteria
WB – White bacteria
GB – Green bacteria

LB
WB
WB

LB/Amp
NB
WB

LB/Amp/Ara
NB
GB

Unit 18 - Assignment 4 - P7
Genetic engineering where an organism’s genetic material is altered or selected, in hope that the
organism will have specific characteristics
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Examples of genetic engineering:
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"Cloning - One of the most controversial uses of genetic engineering has been cloning, or
producing a genetically identical copy of an organism
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Glow-in-the-dark cats - It sounds strange, but in 2007, scientists in South Korea altered the
DNA of a kitty so that its fur would glow in the dark, and then cloned other cats from it,
making the world’s first glowing cats
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Genetic engineering has allowed these plants to be resistant to
certain types of pesticides, so that when the fields are treated to remove pests, the plants
will remain unscathed
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Cows that fart less than average have been produced to fight
the deleterious effects that cow flatulence can have on the environment
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The plants were many times more efficient at cleaning certain
pollutants than regular poplars
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The
result is that people without access to many vitamins will get a healthy dose of vitamin A
when the rice is consumed
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The result is that
manure, which is often made from pig waste, is less destructive to the environment due to
its lower phosphorous content
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Genetic engineering has produced trees that can ward off biological attacks, grow more
quickly and strongly, and create better wood than trees that are not genetically modified
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These are engineered to produce tomatoes that can remain
fresh for longer, can be shipped farther from where they are grown, and can be harvested all
at the same time rather than harvesting only parts of a field at each harvest
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Insecticide corn - Instead of spraying insecticide on plants, why not genetically engineer
crops that kill pests on their own? Corn was developed through genetic engineering to
produce a poison that kills insects
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The banana vaccine - Bananas were developed through genetic modification that offer
vaccine against diseases such as cholera and hepatitis
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"

Reference: http://examples
...
com/examples-of-genetic-engineering
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Examples of
these include: God laid down the structure of creation and any tampering with it is sinful;
manipulating DNA is manipulating 'life itself' - and this is tampering with something that God
did not intend humanity to tamper with
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Examples of these
include: as human beings have been given dominion over the animals, they are entitled to
tamper with them; palaeontology shows that the structure of creation has changed over
time, as some species became extinct and new ones came into being
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Transgenic animals can pose problems for religions that restrict the foods that their
believers can eat, as they may produce animals that appear to be one species, but contain
some elements of another species
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Commercial:
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Biotechnology would be seen as an advantage for animals
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Biotechnology would also be seen as a disadvantage for animals
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For example,
pigs have been bred to grow extra fast, however, some breeds now grow too fast for their
hearts – this results in discomfort when animals are too active
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Profitability is one of the main reasons selective breeding and genetic engineering are
carried on
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This would prevent altering animals in a
harmful way
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The aim is to make a genetically engineered mammal which lacks
the ability to feel, see, hear, smell, or taste, but is otherwise identical to normal
experimental animals
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This is because genetic engineering involves manipulating animals for human advantage
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Recent
action to allow animals to be obtained, reinforces the argument of animals being the
property of humans, and the arguments in favour of animal rights
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The first step of this method
is of course sampling DNA
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Detergent is used to break open the
cells and separates the useable DNA from the extra cellular material
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The
result of this is many fragments (called restriction fragments length polymorphisms) varying in
length, and either blunt of sticky ends being produced
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They are sorted into size using gel
electrophoresis
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This happens as soon as the electric field's current is turned
on
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The DNA
will then become single stranded as the hydrogen bonds will begin to break which causes the
nucleotides to become free (these will be used to pair up with probes; this is due to the alkali
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The fragments are
transferred from the gel, to the surface of the nylon
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The probes will join to any fragments of DNA which share the same
structure
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The probes will leave marks on
the film where they attached to the restriction fragments length polymorphisms
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To be able to match the finger print with other, the x-ray is
places on a light background, and the restriction fragments length polymorphisms lengths in the
DNA are compared from the crime scene, to the DNA of the suspect
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com/slide/5721366/

Genetic fingerprints questions
1- Describe how genetic fingerprinting is carried out
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The length is used to find out how many times the 15-nucleotide sequence is repeated
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The sites and the lengths of the resulting
fragments will vary for each person
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The DNA is made detectable as soon as the
probe binds to the DNA fragment
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If
they are the same length, we can see that they belong to the same person
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The frequency of the differing
DNA patters of different genes will vary; this will of course depend on the population
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2- All three children on the chart had the same parents
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Which of
the other three adults on the chart was the other parent? Give the reason for your answer
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(a) Explain why the polymerase chain reaction was used in this investigation
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(b) In the polymerase chain reaction, DNA is heated to 95 °C and nucleotides, enzymes and DNA
primers are added to the mixture
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(1 mark) To separate the two DNA strands
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(iii) Why are DNA primers added during the polymerase chain reaction? (1 mark) DNA primers are
required for DNA to be replicated
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(iv) What is the advantage of the enzyme used in the polymerase chain reaction being
thermostable? (2 marks) They do not denature in extreme heat like normal enzyme; they are also
able to with stand acidic and alkaline state as well
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hms
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edu/flash/2015/how-to-make-a-gmo/

Stage 1 – Identifying a trait of interest: an example of this stage is, researchers would look for
organisms what are able to survive in a certain environment, if they needed a trait which would
allow a crop to survive in that certain environment
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Stage 2 – Isolating the genetic trait of interest: Comparative analysis is used to identify which part of
an organism's genetic makeup contains the desired trait
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The aim of this is

to find genes that are only present in the former
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If there is
no genetic information to compare, the parts of the desired genome would be deleted from the
database, until the trait of interest is lost; this would therefore identify the genes which leas to the
trait
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Gene guns are therefore used to shoot DNAcoated metal particles into plant tissue
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This is done
by inserting pieces of their own DNA into a plant's genome
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Before this, they must check the genome of the organisms; this is so that
the researchers are only breeding organisms which the genome was correctly modified
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Why was most bacteria plate grown on medium containing Amp (Ampicillin antibiotic)?
When the antibiotic is used, we are able to observe what the plasmid does
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2
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Under a standard environment, the
plasmid will not produce the luminous protein
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This would cause them to grow
at a very slow rate
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3
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This means that it will need to recover

4
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On the other hand, if there was no calcium
chloride in the solution, the negative charge of the DNA would be unprotected
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5
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The reason for this
was that the plate did not have a plasmid, however there was ampicillin in the agar
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