Theeffectofincreasingordecreasingthemassofthesaltaddedina

DNAextractiononthethickness(inmm)oftheDNAextracted
JoshuaLau10J

Aim-
The aim of our experiment is to investigate how increasing or decreasing the mass of salt
added can increase or decrease the quantity of DNA extracted in a dna extraction
experiment. Our approach to this is to extract the DNA from several kiwis four times, keeping
all variables the same each time with the exception of the mass of salt in order to observe
the effect that salt might have on how much DNA will be extracted, which we will quantify by
measuring the thickness, or height, of the DNA extracted within the test tubes.

Research-

1) Why do scientists/forensics extract DNA?

There are a variety of reasons as to why scientists or forensics would make use of
extracting DNA.
Firstly, the discovery of DNA extraction has greatly impacted our society from a
medicinal standpoint. Because of it, we can diagnose diseases with ease, having gained the
ability to properly identify the genes that could potentially trigger said diseases or viruses
(Murnaghan n.pag.). Because of this, we are able to develop drugs that could treat these
diseases, saving the lives and well-being of many. Because of this discovery, scientists have
been able to identify the causes of genetic disorders such as the sickle cell disease and
down syndrome.(Nicholson n.pag.)
Secondly, it has also greatly aided in forensics. From just simple pieces of evidence
in crime scenes such as hair, saliva, blood, or other genetic materials, (A Simplified Guide To
DNA Evidence: How Its Done, n.pag.) forensic scientists can either determine the guilt or
innocence of a person in question, or determine the identity of the victim in the event that
his/her corpse is unrecognizable. (Murnaghan n.pag.)
Lastly, it has also been beneficial to agricultural industries. Firstly, it has allowed
breeders to breed animals with a greater resistance to diseases (Murnaghan n.pag.), not only
benefitting the breeders from a financial standpoint, but also benefitting the consumers from
the aspect of health. In addition, farmers can also produce more nutritious crops with the
help of findings in DNA extractions. Take the banana for instance. The analysis and
modification of the fruit on a genetic level has allowed scientists to increase the Vitamin A in
the fruit, a discovery which could potentially save millions from vitamin A deficiency. (Coming
soon: Genetically edited 'super bananas' and other fruit? n.pag.)

2) Why is Detergent, salt, and alcohol used in DNA extraction and what effect do
they have on the cell?

Detergent: Detergent is used in a DNA extraction in order to break down the fats and
proteins (Extracting DNA from any living thing n.pag.) that form up the cell membranes of the
(in my case) kiwi cells, which then separates the nuclear contents (which holds the DNA)
from other cellular components, and by doing so, releasing the dna cells.

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 Salt: In a DNA extraction, the purpose adding salt to the kiwi mush+water+detergent
solution is to neutralize the negative charges of the DNA molecules. Because of this, the
separate DNA molecules will have a greater chance of collecting together (Classroom
Activities in Plant Biotechnology n.pag.) and becoming visible for the conductors of the
experiment, which can help make the process of analyzing the DNA much easier.

Ethanol: Because the DNA is not visible after being released from the cell nucleus
(since it dissolves in the water+salt+detergent+fruit mush solution), Alcohol, or in our case
ethanol, is used primarily in a DNA extraction to precipitate the DNA out. This is because
DNA is insoluble in ethanol, and will thus cause the DNA proteins to emerge out of the
solution. Additionally, through this, other cellular components (Extracting DNA from any living
thing n.pag.), plus any remaining salt residue are then washed away (Protein Man n.pag),
purifying the DNA extracted and allowing the DNA to be taken out of the solution using a
glass rod for further analyzation.

Hypothesis-
I predict that as we increase the mass of the salt added during the the DNA extraction, the
quantity of DNA extracted (measured in thickness/height) will increase at the same time.
This is because with a greater mass of salt within the (kiwi+detergent+water+salt) solution,
more negative charges in the kiwi DNA molecules will be neutralized, likely resulting in a
greater quantity of DNA molecules collecting together and becoming visible for us. However,
I believe that this positive trend/relationship between the mass of salt and quantity of DNA
extracted can only reach to a certain extent, since there will come a point in which there will
be enough salt for all the negative charges in the DNA molecules to be neutralized, thus
resulting in a
ll the DNA within the solution to emerge together in one clump. (no amount of
salt can add DNA molecules into the solution)

Variables-
Independent Variable: Mass of Salt (Unit of measurement: Grams (g))
Dependent Variable: Height/thickness of DNA extracted (Unit of measurement: mm)
Controlled variables: See table below
Controlled Unit of Reason/justification
Variable meas-
ureme
nt

Volume of ml For each of the 4 times i manipulate the independent variables, the
mashed kiwi volume of the 4 mushed kiwis should be kept constant. This can
not be as simple as using 4 separate kiwis, but instead, i will crush
the 4 kiwis in the same ziploc bag and pour them into 4 separate
beakers evenly so that no beaker has any more or any less volume
of the crushed kiwi. If the kiwis were distributed unevenly in the 4
times i manipulated the IV, there would be either more DNA or less
DNA in each result, causing the final results to not properly reflect
the effect of salt on the quantity of DNA extracted (in terms of

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 height and thickness)

Volume of ml The volume of detergent added into the kiwi mush+salt+water

detergent +detergent solution should be kept constant in each time i
manipulate the IV. If i were to add or decrease the volume of the
detergent, the quantity of DNA extracted would likely add or
decrease accordingly, since increasing the volume of detergent (as
i found out in the research above) would break down more cellular
structures and thus potentially release more DNA into the solution,
and again, causing the final results to not properly reflect the effect
of salt on the quantity of DNA extracted (in terms of height and
thickness)

Volume of ml Adding more or less Ethanol in the experiment can potentially affect
Ethanol the result of the experiment. As i found in my research above, the
role of ethanol in a DNA extraction is to precipitate the DNA out of
the solution. While it is unlikely that changing the volume of the
Ethanol will affect the results (since even if there are a few extra or
less milliliters of ethanol, all the DNA should precipitate out
regardless), I should keep the volume of Ethanol constant to
prevent any other possible unknown factors that could alter my
results. If there is far too little ethanol, we run a risk that the DNA
will not precipitate properly, if there is too much, it could affect the
way it floats and forms after it emerges, thus affecting the results
and not properly reflecting the effect of salt on the quantity of DNA
extracted (in terms of height and thickness)

Temperature Celsius The hot water bath should be kept at a constant temperature of 60
of Water (C) degrees celsius. This is done in the first place because proteins
bath can be further broken down in a higher temperature, purifying the
DNA further. Increasing the temperature too much could cause
even the DNA to dissolve or denature, while a low temperature
could influence the yield of DNA (or quantity of DNA extracted).
Therefore i must keep it at a constant temperature, or else it would
affect the results and poorly reflect the effect of salt on the quantity
of DNA extracted (in terms of height and thickness).

Time in minutes The time the solution should be kept at a constant of 10 minutes. If
water bath i were to leave it in a water bath for too long (15 minutes according
to source), the DNA molecules will begin to break down.
Additionally, if i changed how long i left the solution in the water
bath, the thickness/height of the DNA extracted will be influenced,
since the time the solution is left in the water bath can also dictate
the yield of DNA, and by altering the time left in the water bath i am
changing a variable that is not the IV, which could potentially the
results to poorly reflect the effect of salt on DNA extracted (in terms
of height and thickness)

volume of ml Increasing or decreasing the volume of water will affect the

water concentration of the kiwi+salt+detergent+water solution, so the
volume of the water should remain constant. If the volume of water

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 added is changed, then the solution poured into the test tubes will
posses a lower concentration, meaning there will be either less or
more (depending on whether water is added or reduced
respectively) DNA in the solution, which will affect the result,
causing the results to not solely reflect the effect of salt on the
quantity of the DNA extracted (in terms of thickness and height).

Diameter/ra- mm If the diameter or the test tubes was not constant, measuring the
dius of test height and thickness of the DNA would not accurately show how
tube much DNA was extracted. This is because the only instance where
using thickness to quantify the DNA extracted makes sense is
under the circumstance that the diameter of the test tube remains
constant and thus only changing variable in the equation which
calculates volume, is h (or height). If i were to use tests tubes of
different diameters then i would have to calculate the volume of the
DNA instead for it to make sense.

Equipment list-
4x Cheese cloth
8x 300ml beaker
1x Ziploc Bag
1x Knife
1x Spoon
1x Weighing scale
1x Water bath at 60 degrees celcius
1x Funnel
12x test tube
3xDroppers
1x bottle of salt
1x bottle of Detergent
1x jar of Iced ethanol
4 kiwis (they will be distributed evenly in the method so the fact that the 4
kiwis have different weights is irrelevant)

Risk Assessment-

Source of Risk Method of prevention

hazard

Ethanol -Ethanol is highly To prevent the ignition of ethanol, we

flammable, so will use droppers to add the ethanol to
ethanol ignition is a the solutions
potential risk
Keep in mind to avoid unnecessary skin
-Entering Eyes contact

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 Keeped sealed when not in use to
prevent explosions

Wear protective eye goggles

Knife -Running the risk of Keep blade of knife pointing away from
cutting yourself or any of your body parts and cut in
others when peeling direction away from yourself or anyone
the kiwi
Do not walk around with knife

Kiwi -Affecting people who -before conducting of experiment ask

are allergic to kiwi whether anyone is allergic to kiwi. If so,
inform supervisor or teacher

Beaker/test -Broken glass, cutting -keep such lab equipment away from
tube/glass rod/ skin sides of table and in the centre to avoid
it being knocked off accidentally

-be careful and hold such lab equipment

with a firm grip

-Wear protective lab equipment to

minimize chance of being cut

-in the event of broken glass inform

supervisor or teacher immediately to
prevent others from accidentally
stepping on the shards and hurting
him/herself

Detergent -Irritable to skin -wear protective lab equipment at all

times to prevent unnecessary contact
with the substance

-if in contact with skin wash immediately

Method-
1. Take out innards of 4 kiwis using a knife and spoon and place them into a ziploc bag
2. Make sure there is no air in the ziploc bag, then proceed to crush the kiwis for 5-7
minutes
3. Pour the crushed kiwis into four 250 ml beakers, distributing them in even quantities,
then label each beaker as: 1g, 2g, 4g, and 6g respectively
4. Using a separate beaker, add 80mls of water, then place it on a scale and reset it to
0 grams
5. Using the scale to measure, add exactly 1 gram of salt into the beaker

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 6. Stir until the salt dissolves, then add 10 ml of detergent and mix gently to prevent
bubbles
7. Add the solution made in steps 4-6 into the beaker labelled 1g (from step 4) then mix
gently for 30 seconds
8. Repeat steps 4-7 three times, but instead using 2g, 4g, and 6 grams of salt instead of
1 gram in step 5, and adding the solution to the respectively labelled beakers in step
7 instead
9. Place the solutions into a hot water bath with a temperature of 60C for 10 minutes
10. After 10 minutes have passed, take the solutions out of the water bath
11. Pour the solution labelled 1g through a cheesecloth and funnel into an unused
beaker and label the unused beaker 1g final to avoid confusion. Repeat this step
with the solutions labelled 2g, 4g and 6g, labelling them 2g final, 4g final, and 6g
final respectively
12. Take the solution labelled 1g final and pour it into 3 test tubes until full and label
them 1g-test 1, 1g-test 2, and 1g-test 3 respectively
13. Using droppers, iced ethanol to the 3 test tubes until there is a layer with a height of 2
mm into the test tube and make sure the solution and iced ethanol do not mix
14. Wait for 5 minutes to let the DNA precipitate out fully, then proceed to use a ruler to
measure the height/thickness of the extracted DNA in the three test tubes, and
record it down in a table
15. Repeat steps 12-14, but instead using the solutions labelled 2g final, 4g final, and
6g final in step 12 instead

Results-

Table showing the thickness of the DNA extracted with different masses of salt
added to the solution
Test 1 Test 2 Test 3 Average

1g of salt 12mm 12mm 10mm 11.33mm

2g of salt 7mm 6mm 7mm 6.66mm

4g of salt 12mm 10mm 11mm 11mm

6g of salt 9mm 13mm 12mm 11.13mm

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Graph-

Discussion of results (helps understand the conclusion)-

Through analyzing the graph above, we can see that if we were to take into account all of
our results, our experiment would have yielded an inconclusive result, and failed to display
any relationship between the mass of salt added on the thickness of the DNA extracted.
However, if we were to regard the results with 2 grams of salt as a stray result, then we
would be presented with a fairly conclusive conclusion, which is: the mass of the salt added
(in the region from 1g(x)g6g) does not influence the quantity of DNA (measured in
thickness/height) extracted in a DNA extraction. To further support this conclusion, we also
have fair reason to disregard the results with 2 grams of salt, which is the fact that when
we conducted the portion of the assessment where the independent variable was at a value
of 2 grams of salt, we did not follow the method thoroughly, namely as to how we did not
follow step 7 properly. We were supposed to pour the water+salt+detergent solution into the
kiwi mush, which we had followed properly when using 1g,4g, and 8g of salt. Instead, we did

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it the other way around and poured the kiwi mush into the water+salt+detergent solution.
Because we only gently mixed it for 30 seconds to prevent bubbles, that meant the solution
with 2g of salt probably had a distribution different to the solutions with 1g, 4g, and 8g of salt
respectively, and was likely the cause of the inconsistency in the final results. With reason
to disregard the results with 2 grams of salt, i created a graph with line of best fit
between 1g and 4g of salt, which would likely reflect the result had we properly followed
the method, which is shown below

In this revamped version of the final results, there is only a disparity of 0.33 millimeters,
or 330 micrometers, between the highest and lowest results, giving us a far more conclusive
result when compared to the disparity of 1.7 millimeters, or 1700 micrometers which would
have had been the case had i not drawn a line of best fit and disregarded the stray result.

Conclusion-

In my hypothesis, I predicted that as the mass of salt increased, the quantity of DNA
(measured in thickness/height) would also increase at the same time. However, as seen in
the results and graph above, my hypothesis was incorrect. Despite this, I believe that the
reason as to why my hypothesis was incorrect is also located within my hypothesis. In my
hypothesis, i stated that the positive relationship between the mass of salt and quantity of
DNA extracted (measured in thickness/height) can only reach to a certain extent, which was
when there was enough salt for all the DNA within the solution to emerge together in one
clump. So what does this have to do with my results? I theorize that the reason as to why
there was little to no variation in the results when i changed the independent variable, and
why the results stayed around the region of 11 millimeters (as stated in discussion, the results with 2
grams of salt was ultimately disregarded, since when we conducted the section of the experiment with 2 grams of salt we made
a fairly large mistake and by doing so failed to keep the method constant across the 4 times we manipulated the independent
variable, which was likely the cause of the inconsistent result) was
because this extent of the positive
relationship between the mass of salt and the quantity of DNA extracted (in terms of
thickness/height) that i mentioned in the hypothesis was already below 1 grams of salt, our

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lowest variation of our independent variable, meaning all our results already had the
maximum possible DNA extracted from the solution, and explaining why there was little to no
variation in the final results. With this in mind, i theorize that if i were to use lower masses of
salt when changing the independent variable, i would be able to observe the positive
relationship between the mass of salt and the quantity of DNA extracted (in terms of
thickness/height).

Evaluation-

From analyzing our results, I would say that our method was quite successful and accurate.
Apart from the results with 2 grams of salt, (which was, as i elaborated on in the discussion, purely due to a
misunderstanding of the method, and not a problem with the method itself) our results were very concise with a
disparity of only 0.33 millilitres, or 330 micrometers, properly reflecting how salt would have
no affect on the quantity of DNA extracted after reaching a certain point, in which all the
possible DNA would be extracted. I think that we came up with such an accurate result
because our method allowed us to do all the variations of the independent variable at the
same time, instead of doing one variation of the IV after another. For example, they 4
solutions that had different masses of salt were added to the 4 beakers with kiwi mush at the
same time, or how the 4 solutions took the water bath together. Not only did this method
save time, allowing us more time to do more tests, it also minimized the chance of outside
factors affecting the results in different ways, because we were monitoring all 4 at the same
time, making it harder for one to be effected without the others being too.

Evaluation- suggested improvements

Despite the accuracy of the results, there are still improvements to be made to the method.
The main problem is that i think changing the independent variables only 4 times is not
enough to acquire a conclusive result. If i were to manipulate the independent variable
several more times, the conclusion would more accurately reflect the actual relationship
between the mass of salt added and the quantity of DNA extracted (in terms of height and
thickness). As stated in my conclusion, If i were to have smaller masses of salt, i would likely
be able to find the positive relationship between the mass of salt and the quantity of DNA
extracted (in terms of height and thickness). If i added those variations of the IV into the
graph, i would not only identify the positive relationship between the mass of salt and
quantity of DNA extracted (in terms of height and thickness), i would also be able to locate
the point in which there is enough salt for the all the DNA to precipitate out. The reason as to
why i did not manipulate the independent variable too much was because due to time
constraints and fear of not being able to complete the assessment within the 2 periods that
were offered to us, but looking back i am certain that we played it too safe and could have
had room for at least 2 more variations of the independent variable.

In addition, there are several problems with our method of quantifying the DNA extracted.
Our method was to quantify it by measuring the height, or thickness, of the DNA extracted
inside the test tube. Even though we tried to make it accurate by keeping all the test tubes at
the same size, and by pouring the solution and the ethanol evenly in each test tube, the

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method of measuring the height in the first place was flawed. Firstly, when the DNA floated
out, it was almost never in a fully cylindrical shape inside the test tube. Sometimes the DNA
would have more DNA on one side than the other, making it hard to measure with a ruler,
and forcing us to make an informed guess as to the average height of the entire piece of
DNA. And because some pieces of DNA spread out to the edges of the test tube it was in,
while others did not, the measurements we calculated might not have had been fully
representative of the actual quantity of DNA that was extracted.

Bibliography

Murnaghan, Ian. "The Importance of DNA." The Importance of DNA. N.p., 21 Sept. 2016. Web.
25 Sept. 2016.

Nicholson, David. "15 Ways Our Understanding of DNA Changed the World."LinkedIn. N.p., 20
Nov. 2014. Web. 25 Sept. 2016.

Uncredited. "A Simplified Guide To DNA Evidence: How Its Done" Forensic Sciences Simplified.
National Forensic Science Technology Center, n.d. Web. 25 Sept. 2016.

Cell Press. "Coming soon: Genetically edited 'super bananas' and other fruit?." ScienceDaily.
ScienceDaily, 13 August 2014. Web. 25 Sept. 2016.

Man, Protein. "Work of Salt, Isopropanol and Ethanol in DNA Extraction."G-Biosciences.

G-Biosciences, 7 Sept. 2012. Web. 25 Sept. 2016.

Uncredited. "Extracting DNA from any living thing." Biology Junction. N.p., n.d. Web. 25 Sept.
2016.

Uncredited Classroom Activities in Plant Biotechnology The American Phytopathological

Society. The American Phytopathological Society, n.d. Web. 25 Sept 2016.

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