Y10 Biology Lab Report

Name: Katii Tang

Grade: Y10 Hope
Teacher: Mr. Kitwood

Experiment to investigate the effect of the mass of salt on DNA extraction

Investigation Question
To what extent does the mass of salt (g) affect the extraction of DNA precipitate (cm)?

Background to the Investigation Question

Why scientists/forensic scientists extract DNA
DNA, or deoxyribonucleic acid[1], is a long molecule that contains our genetic code[3]. These codes dictate the
many characteristics in humans and almost all other organisms[2]. The discovery and our understanding of its
functions was argued as the most important discovery within the last century[4]. DNA has had a significant effect
on scientific and medical processes. The effect of the discovery of DNA on scientific and medical progress has
been enormous[4]. For example, scientists can extract DNA at an early step in many diagnostic processes which is
used to detect bacteria or viruses in the environment, diagnose diseases and also genetic disorders[5]. As a result,
drugs could be developed to treat certain diseases and has saved manys lives, which has also led to a
breakthrough in drugs and treatments for patients diagnosed with serious diseases[4]. Scientists were also able to
identify the causes of many disorders, such as down syndrome and sickle cell disease, , by developing an
understanding on DNA[12]. Moreover, due to the uniqueness of DNA which has the ability to identify different
individuals, it is used by forensic investigators in crime science, by taking samples of hair, blood, fingerprints and
etc. Laboratory technicians can then easily identify the criminals involved in the crime[6]. DNA has revolutionised
criminal investigation and helped forensic scientists identify criminals and victims easily[12]. DNA is also pivotal
in agriculture, by using it as a tool to modify important species of crop, in order to boost crop yields and decrease
the chance of insects and diseases[6].

Why is detergent, salt and alcohol used in DNA extraction?

Detergent: The DNA which we need to extract is inside the cell nucleus of the strawberries, and the membranes
would have to be broken down in order to free the DNA[7]. These cell walls are made of fat, and detergent is used
in DNA extraction to break down the fats and proteins which makes up those cell walls which surrounds the cell
and nucleus[7]. The DNA can then be extracted after the membrane is broken apart[7].
Salt: Salt is used to solidify and let the DNA to be visible to the conductors of the experiment as a clump[8]. It
neutralises the charge of sugar phosphate backbone of the DNA, thus enabling the DNA strands to collect
together[9]. Adding salt can also help the DNA strand to dissociate from the histone protein[11], in which the DNA
is wrapped.
Alcohol: DNA itself is soluble in water, as the sugar and phosphate molecules that forms the DNA backbone are
hydrophilic(dissolves in water)[10]. Alcohol pulls water away from the DNA molecule and forces the DNA to
precipitate so it becomes visible[9]. With the help of salt, it prevents the DNA from dissolving in water[10].
Because DNA is hydrophilic, they easily bond with water molecules. Salt and alcohol interacts with the DNA
molecules and makes it less hydrophilic[10].

Hypothesis
I predict that as the mass of salt added to the solution (mashed strawberry+water+detergent+salt) is increased, the
thickness of the DNA precipitate extracted would also increase. This is because when salt is added, the charges
on the DNA is neutralised[9] thus enabling the DNA to clump together, and the proteins called histones, which the
DNA is wrapped with, would be eliminated[11]. Because of this, it would be likely that an increase of salt would
result in a larger quantity of DNA precipitate being seen and extracted. However, the amount of DNA precipitate
extracted could only reach a certain extent, because there would come a point where a certain amount of salt
added is enough to be able to neutralise all charges of DNA and eliminate all protein wrapped around it, thus
extracting all of the DNA from the fruit solution, and adding more salt would not add more DNA molecules into
the fruit solution.

Variables
Independent Variable Mass of salt
Unit(s) of IV Grams (g)

Range of IV measured 0g, 2g, 4g, 6g, 8g

Describe and explain The independent variable in this experiment is salt, and it will be changed 5 times in
the procedure to the experiment. An electronic balance would be used to measure 4 groups of salt, 2g,
change the 4g, 6g, and 8g respectively. The independent variable will be changed by putting each
independent variable group separately (one group will have no salt added) into respective beakers filled
with the same amount of mashed fruit, water and detergent. After being put into the
water bath, the thickness of DNA precipitate would be measured, recorded, and
compared. This would be repeated for five trials and the average thickness of the
DNA precipitate for each independent variable will be calculated.

Dependent Variable Thickness of DNA precipitate extracted

Unit(s) of DV Centimeters (cm)

Describe and explain The experiment will be conducted for 5 trials for each independent variable. After
the procedure to going through the same procedure and the DNA has formed, a ruler will be held next
calculate the to the test tube, and the thickness of the DNA will be measured, which is visible
dependent variable inside the test tube.

Controlled Explain why it matters to the Procedure to control it

Variable investigation

Describe and explain Mass of The mass of strawberry is controlled The mass of strawberries
the procedure to mashed because we assume that the same mass of would be measured to
control other variables strawberries strawberry would yield the same amount exactly 30g each time
in the investigation (g) of DNA, so this guarantees that the using an electronic
thickness of DNA precipitate is balance.
comparable as the independent variable
(salt) is changed.

Mass of The mass of detergent has to be The mass of detergent

detergent (g) controlled because this could affect the would be measured to
thickness of DNA precipitate formed. As exactly 10g each time
seen in the research above, increasing the using an electronic
mass detergent would potentially break balance.
down more of the cellular structure[7],
thus releasing more DNA, and the final
result would not accurately reflect on the
effects of my IV (mass of salt) on the
thickness of DNA precipitate extracted.

Volume of ice The volume of iced ethanol has to be The volume of ethanol is
cold ethanol controlled because this could affect the controlled by making
(cm) thickness of DNA precipitate formed. As sure that it lays 3 cm
seen in the research above, ethanol pulls above the fruit slurry in
away water and aids the DNA to be the test tube for each
visible. If too much ethanol is added, too experiment.
much water might be pulled away from
 the DNA[9], or the DNA might not clump
together and we might not be able to see
it with the naked eye[10]; if too little
ethanol is added, the DNA might just
dissolve into the solution, therefore
affecting my investigation on the mass of
salt on thickness of DNA precipitate
extracted.

Volume of The volume of water added to the fruit The volume of water
water (ml) solution should remain the same, because should be measured
adding more or less water would mean accurately each time
that the solution is either more using a measuring
concentrated or less concentrated; and in cylinder.
the end, when the solution is poured into
the test tubes and alcohol is added for the
DNA to form, the amount of DNA
extracted will differ depending on the
concentration of the fruit solution, which
would affect my investigation on the
effect of the mass of salt on the thickness
of DNA precipitate.

Concentration The concentration of ethanol added to the The same bottle of 96%
of ethanol (%) test tube has to be controlled because the ethanol should be used
it could affect the way the DNA throughout the whole
precipitates and emerges to the surface of experiment.
the ethanol. This could affect the amount
of DNA to be seen by the conductors of
the experiment, thus poorly reflecting on
the effect of the mass of salt on the
thickness of DNA precipitate extracted.

Temperature The water bath should be kept at 60oC A water bath of 60oC
of water bath throughout the experiment. This is should be used
(oC) because in the hot water bath further throughout the whole
isolates the DNA from its cells, and experiment.
proteins can be further broken down,
purifying the DNA. If the temperature is
increased, the DNA might dissolve or
become destroyed, while a low
temperature might not be effective in
isolating the DNA, thus reducing the
amount of DNA extracted in the end.
This would not properly reflect on the
effect on the mass of salt on the thickness
of DNA precipitate extracted.

Time in water The duration that the solution is kept in A timer should be set to
bath (mins) the water bath should be kept to 10 10 minutes to make sure
 minutes. This is because the proteins are the solution is out of the
continuously broken down in the water water bath at 10 minutes.
bath. If the DNA is left in the water bath
for too long, it might be denatured. Also,
changing the duration of the DNA in the
water bath would affect the yield of
DNA, so changing the time would poorly
affect my investigation on the effects of
the mass of salt on the thickness of DNA
precipitate extracted.

Amount of The amount of fruit slurry in the test tube The fruit slurry should
fruit slurry in for adding ethanol has to be the same, be filled of the way
test tube because the more fruit slurry there is, the full in each test tube.
more DNA there will be extracted
because there is more of the strawberries
in the test tube. If each test tube had a
different amount of fruit solution, it
would poorly reflect on my investigation
on the effects of mass of salt on the
thickness of DNA precipitate extracted.

Materials
Strawberries x 35
Detergent x 250g
Salt x 50g
96% Iced Ethanol x 1 bottle
Water x 500ml
Zip-lock bag x 1
Cheese cloth x 1
Glass rod x 1
Knife x 1
Tile x 1
Electronic balance x 1
Water bath at 60oC x 1
Spatula x 1
Dropper x 1
Measuring cylinder x 1
Ruler x 1
Test tube marker x 1
Test tubes x 5
Test tube rack x 1
100ml beaker x 5

Risk Assessment
Safety Reason to consider Prevention/Treatment
Issue the safety issue

Detergent - Could cause skin - Keep lab coat and safety goggles on at all times to prevent spillage on
 irritation and skin.
redness. - Prevent unnecessary contact with the substance.
- Irritates the eyes. Treatment: Wash off with water immediately when in contact.

Ethanol - Highly flammable - Keep lab coat and safety goggles on at all times to prevent spillage on
and has risk of skin.
ignition. - Prevent unnecessary contact with the substance.
- Exposure to skin - Keep bottle sealed when not in use to prevent spillage or explosion.
and eye can cause - Know location of fire extinguisher.
burning and Treatment: Wash with lukewarm water for 10-15 minutes and seek medical
stinging. advice.

Knife - Risk of cuts and - Make sure to cut downwards and away from the body.
injuries when - Cut on a stable surface.
cutting fruits. - Keep knife tip down when walking around.
Treatment: Cleanse with water and use direct pressure to stop the bleeding.
Seek medical advice.

Glassware - Broken glass might
cause injuries.
- Glassware should be transported carefully with a firm grip.
- Always place glassware at the centre of the table to prevent from
knocking.
- Do not run with glassware.
Treatment: Report to the teacher immediately in case of any glass
breakages.

Strawberr - Might trigger - Keep students who have allergies towards strawberries away from such
ies allergies experiment.
Treatment: Report to the teacher immediately in case of allergy and seek
medical advice.

Method
1. Cut out stems of all strawberries and place them into the Ziplock bag. Release any air from the
bag, and crush the strawberries until there are no visible lumps.
2. Using an electronic balance, measure 10g of detergent, 8g of salt, and 30g of mashed fruit into a
100ml beaker.
3. Reset the scale to 0 grams, and add in exactly 8g of salt into the same beaker.
4. Reset the scale to 0 grams, and add in exactly 30g of the prepared mashed strawberries into the
same beaker.
5. In a measuring cylinder, measure out 20 ml of water, and add it into the same beaker.
6. Using a glass rod, stir the mixture until no solids are visible.
7. Place the prepared beaker into a 60oC water bath for 10 minutes.
8. After 10 minutes, remove the beakers from the water bath. Remove and pass the solution through
a piece of cheesecloth into another beaker.
9. Add the filtrate to a test tube, until it is filled up of the way full.
10. Slightly tilt the test tube; using a dropper, slowly add the iced ethanol until there is 3 cm above
the fruit slurry. Keep the ethanol as separated with the fruit mixture as possible,
11. Let the test tube sit on a test tube rack for 5 minutes, until the DNA is fully precipitated.
 12. After 5 minutes, use a ruler to measure the thickness/height of the DNA precipitate visible in the
test tube, and record it down on a table.
13. Repeat steps 2 to 12 for 4 more times, so a total of 5 trials are recorded.
14. Repeat steps 2 to 13 for the other independent variables, by adding 6g, 4g, 2g and 0g of salt.

Results
The effect of the mass of the salt added in a DNA extraction and the thickness of DNA precipitate extracted
Thickness of DNA precipitate (cm)
Mass of salt (g)
Test 1 Test 2 Test 3 Test 4 Test 5 Average

0 0.2 0.3 0.4 0.2 0.4 0.30

2 2 1.8 1.2 1.7 0.9 1.68

4 1.5 1.4 1.7 1.6 1.7 1.58

6 1.4 1.5 1.7 1.8 1.7 1.62

8 1.6 1.7 1.6 0.7 1.6 1.63

*Red: Outliers
Graph
Qualitative Observations

After the ice cold ethanol has been poured into the fruit solution, the two
layers will be separated. The DNA precipitate will form and float near the
surface of the ethanol as a thin ball of white fiber.

Conclusion
According to the findings above, my results supported my predictions, which proves the validity of my
hypothesis. The amount of DNA precipitate extract has increased as more salt is added, until a certain extent
where all of the DNA present in the fruit solution is extracted out completely, and thus the results leveled off at a
point of the graph. When no salt was added to the solution (fruit+detergent+water), the average DNA precipitate
extracted for the five trials was 0.3cm thick; when 2g of salt was added, the average DNA precipitate extracted
amongst the five trials was 1.68cm thick; and when 4g, 6g and 8g of salt was added to the solution, the average
DNA precipitate extracted for the five trials were 1.58cm, 1.62cm, and 1.63cm thick respectively. It can be seen
that by increasing the salt amount added to the solution from 0g to 2g, the DNA precipitate extracted has
increased by 1.38cm; but from 2g until 8g of salt was added to the solution, there was only a disparity of 0.1cm
(1mm) between the highest and lowest result.

Explanation of Results
As seen above, as the mass of salt added to the solution increased, the thickness of DNA precipitate also
increased, until an extent where the maximum DNA precipitate that has been extracted out was 1.68cm thick,
which was when 2g of salt was added to the solution (fruit+detergent+water+salt), and the results only had slight
variations afterwards for the other independent variables (Xg>2g of salt); there was only 1mm difference in the
DNA precipitate extracted between highest and lowest results. According to my research, I theorize that the
reason to as to why there was only a very slight variation between the results after the mass of salt was increased
onwards at 2g, was because the positive relationship between the mass of salt added to the solution and the
amount of DNA precipitate extracted can only be seen below 2g of salt; the charge of the sugar phosphate
backbone of DNA[9] was neutralised and the dissociation of the DNA strand from the histone protein[11] could
already be fully completed when a certain amount of salt is added under 2g for this sample amount of
strawberries. This means that after 2g of salt is added to the solution, all of the DNA from the sampled fruit
solution has already been fully extracted out, which shows why there is only slight difference between the final
results of independent variables with the salt amount added that was above 2g. I predict that if I were to choose a
different set of independent variables with lower masses of salt added to the solution (i.e. 0.3g, 0.6g, 0.9g), it
would better show the positive relationship between the mass of salt and its effect on the thickness of DNA
precipitate being extracted.

Evaluation - Validity
From my findings above, I would say that our method was successful and appropriate to investigate our research
questions. In the end, we have concluded that results after the adding 2g salt (IV), it has reached the maximum
amount of DNA precipitate that could be extracted from a fixed amount of fruit sample (in this study, around 6g
of fruit tissue sample was used in each investigation, as 30g of mashed fruit+detergent+salt+water was divided
equally amongst 5 test tubes), and any additional salt added would not have an effect on the thickness of DNA
precipitate extracted, and thus all independent variables that was greater than 2g (salt) only had a 1mm difference
between the highest and lowest results. Our method was valid because it has been scientifically proven to be used
to extract DNA from fruits, by squeezing the strawberries to release the contents in the cell, and free the DNA[13];
then, the addition of detergent would aid the process of the cell to lyse so that the DNA could be released[13]; next,
the addition of salt forces the DNA out of the solution[13]; and finally, because the DNA is insoluble in alcohol,
the addition of iced ethanol helps the DNA precipitate and become visible to the conductors of the experiment[13].
Moreover, our method has allowed us to achieve more accurate results by having conducted 5 trials for each
independent variable, which reduced the chance of any random errors that were uncontrollable, and also helped
identify outliers when we encountered systematic errors in the process when we could compare 5 results with
each other, for example, when there was close to a 1 cm difference between one trial and the other trials.

Evaluation of the Method and Suggested Improvements

State how to improve Explain why the improvements would benefit your investigation
the method

Adding pineapple Salt can promote the dissociation between the DNA and the histone protein. However,
juice into the sample. only adding salt may not completely remove the tightly bounded protein from the
DNA[11]. Pineapple juice contains enzymes that could break down those proteins[11], so
that it could give a more purified DNA extraction.

Measure the mass When measuring the DNA precipitate from the test tube, there might be bubbles
instead of the trapped between the sample, which would affect the measured thickness of DNA
thickness of DNA precipitate. Moreover, the DNA was circled around the test tube, which makes it
precipitate extracted. inaccurate to measure solely by placing a ruler next to the test tube. By extracting and
drying out the DNA precipitate, then putting it on an electronic balance and recording
the mass, would demonstrate a more accurate result for the amount of DNA extracted
from the set amount of fruit tissue sample.

Changing a set of As demonstrated in my findings above, it can be seen that when the mass of salt added
independent to the solution is greater than 2g (Xg>2g), it failed to demonstrate a positive
variables relationship between the mass of salt added to the solution and the thickness of DNA
(0g<Xg>2g). precipitate extracted. By changing a set of independent variables with amount of salt
added as 0g<Xg>2g, I would be able to locate the point in which that that amount of
salt is enough to for all of the DNA to form and precipitate out.

Controlling the Although the amount of fruit sample added in our test tube was one of the controlled
amount of fruit variables (we mentioned to fill the test tube up of the way full), it was difficult to
sample in each test eyeball and evenly distribute the amount of fruit poured into each test tube, and thus
tube by using a ending up with slight differences between the amount of fruit sample added into each
measuring cylinder. separate test tube, and this has resulted in a different amount of DNA extracted with the
same independent variable. By using a measuring cylinder, we could accurately
measure how much of the fruit solution was poured in each test tube and make sure
they are even, and this would result in more evenly distributed results and reduce the
chance of having outliers in our data.

Extensions to the experiment

A possible extension to the experiment would be to test the same method by using other fruits, such as Kiwis or
Bananas, to see whether the same results could be obtained when changing the mass of salt added to the fruit
solution. This would better prove the validity of my results and show that my results were accurate for all given
circumstances.
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