DNA Extraction and Gel Electrophoresis

INTRODUCTION

DNA extraction and separation by agarose gel electrophoresis is a simple and exciting process that
anyone can perform. However, the high cost of specialized equipment and chemicals often hinder such an
experiment from being carried by high school students. Here, we describe a cost effective way of extracting
and electrophoresing DNA using products found in local grocery stores.

The first step in obtaining DNA involves breaking open the cell’s membrane by using physical or
chemical means. For example, you could use ultrasonic waves or vibrations (sonification). Alternatively,
our method involves homogenization using a mortal and pestle and detergent to disrupt the cells’
membrane. Why do people want to obtain genomic DNA? There are a number of reasons. For example,
this DNA could be used to clone new genes or look for special regions of interest. You could also obtain a
number of different genomic DNA and make comparisons with them to identify genetic diseases.

Electrophoresis is a way of separating molecules based on charge and size. In our case, we want to
separate different sizes of genomic DNA molecules obtained from fruits, vegetables and yeast. Generally,
agarose (polysacchardie polymer) or acrylamide (neural toxin) is used to form electrophoresis gels. At the
currect concentration, both types of gels contain appropriate sized pores which allow molecules such as
DNA to pass through. Current is then applied to push the molecules through. Because DNA is negatively
charged, it will travel towards the positive electrode when placed in an electric field. We used agar agar as
our matrix support to resolve DNA as it is cheaper than agarose and safer than acrylamide.

Upon completion of electrophoresis, the location of the bands need to be visualized. A common
way to detect the bands is to stain the gel with ethidium bromide, using ultraviolet light to view the DNA.
Unfortunately, both ethidium bromide and ultraviolet light are carcinogenic and are therefore, not ideal for
use among high school students. We suggest using methylene blue to visualize the bands. This stain gives
good results under suitable staining and destaining conditions.

MATERIALS AND METHODS

General DNA Extraction Procedure

1. Slice up DNA source of choice (fruit, vegetable or yeast).
2. Using a mortar and pestle, grind up about 30 mL DNA while gradually adding 10mL of prepared
detergent buffer solution. Grind for at least 5 minutes with all of your weight and strength to ensure that
you break open the cell membrane and reach a creamy soup consistency. If the sample is too thick after
grinding, add more saline solution to achieve the optimal thickness so that the liquid portion of the
sample is able to pass through the filter, while the larger cellular material remains behind on the filter. If
the DNA sample is frozen, it is considerably easier to grind.
3. Filter your sample’s juice through a coffee filter into a small beaker. You can also use 3-4 layers of
cheesecloth instead of coffee filters. Coffee filters, however, are better because they are cheaper, more
accessible, and easier to cleanup. Let the solution drip into the beaker until all of the liquid has passed
through the filter. If this takes too long, simply squeeze all of the juice from the sample through the
filter.
4. Gradually add ice cold 99% propanol by drizzling it down the side of the beaker. The isopropanol will
form a clear layer on top of your sample. Add 2 volumes (this means approximately two times the
volume of the sample present) of ice cold 99% isopropanol down the side of the beaker with a straw or
pasteur pipette. It is helpful to tilt the beaker as you do this to increase the surface area of the juice
layer in contact with the alcohol. Also, do this step slowly to enable the alcohol to form a layer on top
of the juice layer. If the alcohol does not form a separate layer, the alcohol will be too dilute to
precipitate the DNA. As you let the beaker sit, the DNA should precipitate. The longer you wait, the
more DNA you should see.
5. DNA precipitates, resembling a thread of translucent white snot, at the interface between the juice and
alcohol. After a considerable amount of time, the DNA may eventually float to the top of the alcohol
layer.
Note: If the DNA does not clump together, students can draw up the solution with a pasteur pipette or
an eye dropper. They could then transfer the DNA to a new test tube and continue the normal
procedure. If you have access to a centrifuge, you could spin down the precipitate before pouring out
the isopropanol.
6. Remove the DNA with a wooden popsicle stick or glass rod. DNA adheres well to the wooden stick.
Ideally, you’ll have 0.02 to 0.10 mL of DNA.
7. Transfer the DNA into a clean test tube.
8. Rinse the DNA with 70% ethanol to remove excess salts. Decant the ethanol (i.e. pour off the liquid
portion).
9. Dissolve your DNA into 0.5mL to 1mL of distilled water for about an hour. An alternate way to increase
the concentration of dissolved DNA is to place the DNA smaple with the added 0.5 mL to 1 mL
distilled water into a 55oC water bath for 20-30 minutes.
10. Take about 85uL of DNA sample and add it to a new test tube. Also add 15uL of stop loading buffer to
the sample.
11. Load the above sample (100uL) into the gel covered in electrophoresis buffer.
12. Run sample at 85V for about 1 hour.
13. Stain with 0.02% methylene blue dye overnight.
14. Visualize DNA bands!

In step 2, a variety of different buffers or method can be used to break open the cells’ membrane. The
recipe for each buffer are summarized below in table 1.

Table 1. Buffer recipe / Method used for disturbing cell membranes

Method Recipe / Instructions
Saline Method 0.9% saline
Following step 3, add 1.5mL of 10% dish washing liquid and swirl
gently.
We recommend using Palmolive brand in the unconcentrated clear form.
Regular Buffer Method 1.5g table salt
5.0g baking soda
5mL dish soap (clear Palmolive)
Add distilled water into the container until it reaches a volume of 120mL
Acidic Buffer Method 1.5g table salt
5.0g baking soda
5mL dish soap (clear Palmolive)
Add distilled water into the container until it reaches a volume of 120mL
5mL of vinegar
Basic Buffer Method 1.5g table salt
5.0g baking soda
5mL dish soap (clear Palmolive)
Add distilled water into the container until it reaches a volume of 120mL
5mL of vinegar
10mL 0.4M NaOH
Proteinase Method 1.5g table salt
5.0g baking soda
5mL dish soap (clear Palmolive)
Add distilled water into the container until it reaches a volume of 120mL
2.5mL Complete eye solution
2 protein tablets (Complete brand)
Meat Tenderizer Method 100mL hot water
(used for 1.5g raw wheat germ 5mL detergent
as DNA source) 3g meat tenderizer
Electrophoresis Box
You will need:
- a 12cm X 16cm X 4cm plastic container
- 20cm-25cm of stainless steel wire (20 guage)
- two 5cm stainless steel screws
- five to seven 9Vbatteries clipped together
- two alligator clips
-
Assembling the electrophoresis box is quite simple. First, take a stainless steel screw and attach a
10 cm wire to the end of it while wrapping it around the bottom threads of the screw. Repeat with the other
screw. Once the wires are firmly attached, place the screws at alternate corners of the plastic container so
that they are diagonal from one another. The wires should rest flushed against the bottom edge of the walls
in the plastic container. Secure the screws with electrical tape.

When you are ready to place your gel into the electrophoresis box, connect your 9V batteries in
series. With an alligator clip, attach the battery’s positive terminal to one of the screws. Using another
alligator clip, attach the battery’s negative terminal to the other screw.

Mold for Casting Gels

To create a mold for electrophoresis gels, use a simple square soap dish approximately
1.5cmx5cmx8cm. Then attach a “comb” near one end of the soap dish to create wells in the gel. A
manicurists toe spacer, popsicle sticks and masking tape can be used together to create a comb. Two
popsicle sticks on both sides of the toe spacer are used to balance the spacers along the side of the soap
dish. Ensure that there is at least 3mm distance between the casting tray and the bottom of the toe spacer to
allow the formation of proper wells. Also, before using the toe spacers, it is recommended to cut them in
order to prepare a 3mm x 8mm well. Line the bottom of your soap dish with saran wrap for easier removal
of gel. When handling the agar agar gel, be extra careful not to break it as it is extremely flmisy.

Gels
To make the gel, it is recommended to use agar agar in the powder form rather than the granular
flakes of agar agar. Ensure that it does not contain any additional ingredients such as glucose as these
ingredients may interfere with the formation of the gel matrix. 1% Agar agar. Agar agar in the noodle form
is also available, but are much more difficult to use. They are messy, need to be cut up and strained after
heating. Weigh out ??grams of agar agar and transfer it to an appropriately sized glass Erlenmeyer flask.
The flask should be large enough so that the agar agar solution forms a 2 cm layer at the base. This is
required to ensure a large surface area in contact with the hotplate and to bring the solution to a boil
quickly. It is recommended to use a hot plate and a stir bar to heat the agar solution evenly. An alternative
is to use a microwave, but with this method one must use a low heat level at short intervals while swirling
the flask in between heating. If this procedure is not carried out carefully, the solution will likely overboil
and create a large mess. If the agar agar solution is not completely dissolved, the undissolved bits of agar
will be stained blue during the staining procedure, making it difficult to view the bands of DNA. Also,
undissolved agar prevents the formation of pores, causing the bands to be faint and smeared. Once
dissolved, the agar agar solution should be poured into the plastic-lined soap dish. Remember to also insert
the toe spacers to form the wells. It will take approximately 20 minutes for the gel to solidify. During this
time do not disturb the gel. An indicator that the gel is ready to be loaded is that there will be resistance
while carefully pulling the toe spacers out of the soap dish. The gel should be poured to a 0.5 cm thickness.
If the gel is too thin, causing it to float, the gel should be gently rubbed against the bottom of the
electrophoresis box to create a static cling. For storage purposes, the wells of agar agar gel are often broken
if immersed in running buffer overnight. It is preferred to wrap the gel in plastic wrap and place it into a
fridge.
DISCUSSION AND RESULTS

Detergent Buffer (To break up cell) and DNA Source

Several different methods of extracting DNA were attempted, including: the saline method, the
buffer method, the acidic buffer method, the basic buffer method, the proteinase method, the meat
tenderizer method, and the heating method. The different types of detergents were tested systematically
against a series of different fruits and vegetables and are summarized in the Table 2.

Both the regular buffer method and acidic buffer method allowed us to obtain large quantities of
DNA. The regular buffer method was effective with onion, split peas, frozen corn, bean sprouts, kiwis and
bananas. The acidic buffer method was especially effective with onion, split peas, frozen corn, yeast, lima
bean bacteria, bean sprouts, and bananas. The basic buffer method and proteinase method also allowed us
to collect DNA. Overall, we recommend the acidic buffer method to extract DNA from variou food
sources.

Running Buffer

The preferred Magyver running buffer was made of: 0.05 g/L salt, volumized to 1 L with distilled
water with a final pH of 7.5 ± 0.5. Baking soda (sodium bicarbonate) was used to change the pH of
distilled water from pH 5 to the recommended pH. A superieur alternative to baking soda is an alkaline
buffer made by Seachem. This product is commonly sold by pet food stores to buffer aquarium water. In
this experiment, it was found the Seachem product demonstrates a superior buffering capacity compared to
baking soda. To obtain the optimal pH approximately 3 g/L of baking soda or 0.48g/L of alkaline buffer
solution is required in the running buffer. Litmus paper and a pH meter were used to determine the pH of
the running buffer.

Modified running buffers were also used but found to be unsuccessful. A buffer made of 0.03 g/L
salt in distilled water resulted in the disappearance of the tracking dye, the ethidium bromide, the standard,
and the samples. A concentration of salt higher than 0.05g/L (we used 0.10 g/L salt) in the running buffer
caused the tracking dye to turn from blue to yellow. As well, the DNA standard and samples were not
visible under UV light with an increased salt concentration.

Table 3. Summary of different running buffers, staining conditions, and their effects.
Gel Baking Alkaline Water Salt Other PH Staining Comment
Soda Buffer (g/L)
(g/L) (g/L)
1.0% --- --- --- --- 1X TBE as --- Stain with 20-30 EtBr bands present.
Agarose RB drops of 5% MB in Clear MB-stained
100mL distilled bands for sample and
water for 3 hr. standard present
Destain 2hr, during (Repeated experiment
which changed three times, achieved
distilled water 3 same results)
times.
1.0% --- --- --- --- 1X TBE as PH8 with MB --- EtBr bands for
Agarose RB. 5 drops before and after sample and standard
of 5% MN gel not visible
in 1liter of electrophoresis
RB
1.2% Agar --- --- --- --- 1X TBE --- --- EtBr bands present
Agar for sample and
standards (Repeated
experiment twice,
achieved same
results)
1.0% Agar --- --- --- --- 1X TBE --- --- EtBr bands present
Agar for sample and
standards (Repeated
experiment twice,
obtained same
results)
1.0% 1 0 Distilled 0.05 10-15 PH 7.5 --- Samples including
Agarose drops/L positive control did
Methylene not show MB bands
Blue
1.0% 0 0 Distilled 0 1X TBE ---- --- Samples including
Agarose with 10-15 positive control did
drops/L not show MB bands
Methylene
Blue
1.0% 1 0 Distilled 0.05 ---- PH 8.0 --- EtBr Bands present
Agarose both times these
conditions used
1.0% 1 0 Distilled 0.05 ---- PH 8.0 --- EtBr Bands present
Agarose but they were not
tight bands
1.1% 1 0 Distilled 0.05 ---- PH 8.0 --- EtBr Bands present
Agar Agar but they were not
tight bands
1.1% Agar 1 0 Distilled 0.05 ---- PH 8.0 --- EtBr Bands present
Agar
1.0% Agar 1 0 Distilled 0.05 ---- PH 8.0 --- Samples including
Agar positive control did
not show EtBr bands.
(Repeated experiment
twice)
1.0% Agar 1 0 Distilled 0.05 10-15 PH 5.0 without --- Samples including
Agar drops/L of MB positive control did
Methylene PH 7.0 with not show EtBr bands.
Blue in gel MB (Repeated experiment
and Running twice)
Buffer
1.0% Agar 4 0 Distilled 0.05 10-15 PH 8.0 --- Samples including
Agar drops/L MB positive control did
only in RB not show EtBr bands.
Experiment repeated,
bands present.
1.0% Agar 3 0 Distilled 0.05 10-15 PH 8.0 --- EtBr Bands present.
Agar drops/L MB (Repeated experiment
in RB twice)
1.2% Agar 3 0 Distilled 0.05 10-15 PH 8.0 --- Samples including
Agar drops/L MB positive control did
only in RB not show EtBr bands.
0.8% Agar 4 0 Distilled 0.05 --- PH 8.0 --- Samples including
Agar positive control did
not show EtBr bands.
1.0% Agar 3 0 Distilled 0.05 --- PH 8.0 --- EtBr Bands present
Agar
1.0% Agar 3 0 Distilled 0.05 10-15 PH 8.0 --- Samples including
Agar drops/L MB positive control did
only in RB not show EtBr bands.
1.2% Agar 0 0 Distilled 0.05 10-15 --- --- Samples including
Agar drops/L MB positive control did
only in RB not show EtBr bands
in 3 of 4 gels. Bands
present in samples
and standard of
fourth gel.
1.0% Agar 0 0 Distilled 0.05 --- --- --- Samples including
Agar positive control did
not show EtBr bands.
0.8% 0 0 Distilled 0.05 1X TBE --- --- Tight EtBr bands
Agarose used to only present in samples
make gel and standard
0.8% 0 0 Distilled 0.05 --- --- --- EtBr Bands present in
Agarose samples and standard.
0.8% Agar 0 0 Distilled 0.05 1X TBE --- --- EtBr Bands present in
Agar used to only samples and
make gel standard.(repeat
results twice)
0.8% Agar 0 0 Distilled 0.10 --- --- --- Sample including
Agar positive control did
not show EtBr bands.
Tracking dye turned
yellow.
0.8% Agar 0 0 distilled 0 1X TBE in --- Old stain with Tight EtBr bands
Agar gel and as unknown present in sample and
RB concentration used standard
for 1hr-did not work
0.8%Agar 5 0 Distilled 0.05 Agar PH 8 --- Tight EtBr bands
agar hydrated present in sample and
overnight standard
0.8%Agar 0 0 Distilled 0.05 0.3mL fo PH 9.5 --- EtBr Band present for
agar 0.4M NaOH standard. Tracking
for RB and dye turned yellow.
to make gel. Running buffer didn’t
Agar bubble
hydrated
overnight
0.8% Agar 4 0 Distilled 0.05 Agar PH 7.5 --- EtBr Bands present
Agar hydrated for both standard and
overnight sample.
0.8%Agar 2 0 tap 0.05 --- PH8.0 --- Gel dark brown
agar before
electrophoresis.
Light brown after run
gel. EtBr Bands
present in samples
and standard
1.0%Agar 4 0 distilled 0.05 --- PH 7.8 --- Samples including
agar positive control did
not show EtBr bands.
Gel dark brown
before
electrophoresis.
Light brown after run
gel Repeat
experiment, achieved
visible EtBr bands for
sample and control
1.0%Agar 1 0 distilled 0.05 --- PH 7.8 --- EtBr Bands present
agar for sample and
standards
1.0% Agar 1 0 tap 0.05 8 drops PH 7.8 --- No EtBr bands
Agar 5%MB in visible for samples
1L of RB and standard (Repeat
experiment three
times, achieved same
results)
1.0% Agar 2 0 tap 0.05 8 drops PH 7.8 --- No EtBr bands
Agar 5%MB in visible for samples
1L of RB and standard
0.8% Agar 1 0 tap 0.05 --- PH7.8 --- Samples including
agar positive control did
not show EtBr bands.
0.8%Agar 1 0 tap 0.05 MB only PH7.7 (with --- Samples including
Agar added to RB and without positive control did
dilute MB in not show EtBr bands.
RB)
1.0% Agar 5 0 Distilled 0.05 --- PH 8 without Stain with 40 drops Bands stained with
Agar MB. PH 9 with of MB in 200mL EtBr visible. Stained
MB distilled water for 30 gel too dark to detect
min. Destain bands. Destain O/N,
overnight but no bands clearly
visible.
1.0% Agar 3 0 Distilled 0.05 No MB in PH 8.0 Stain O/N with Clear bands present
Agar RB agitation with 0.02% after MB staining
MB in distilled (Repeated experiment
water. No and achieved same
destaining required results)
1.0% Agar 3 0 Distilled 0.05 No MB in PH 8.0 Stain O/N with Very faint MB bands
Agar RB agitation with are present. Need a
0.002% MB in darker stain solution
distilled water. No to make bands darker
destaining required (Repeated experiment
and achieved same
results)
1.0% Agar 0 0.48 Distilled 0.05 60µL of 5% PH7.8 Stained with 0.05% Bright bands stained
Agar MB per liter MB for 50 min. with EtBr. (Repeated
of RB Stain was too dark. experiment four
Need less times and achieved
concentrated same results)
staining solution.
1.0% Agar 0 0.48 Distilled 0.05 No MB in PH7.8 Stained with 0.05% Bright bands stained
Agar RB MB for 50 min. with EtBr. (Repeated
Stain was too dark. experiment and
Need less achieved same
concentrated results)
staining solution.
1.0% Agar 3 0 Distilled 0.05 No MB in PH8.0 Stained with Bright bands stained
Agar RB 0.025%MB O/N with EtBr. Clear,
with agitation. distinct bands
detected with MB

Note: -EtBr is Ethidium Bromide, MB is Methylene Blue, O/N is over night and RB is Running Buffer.
-Unless otherwise noted, RB was used to prepare the gel
-Solutions made with Alkaline Buffer by Seachem are buffered at pH 8.

We found 1.0% agar agar gel as the most effective solid matrix support for DNA to travel through.
0.8% agar agar gel also worked well but is very fragile and is easily damaged. Below is an additional table
summarizing various combinations of gels and running buffer.

Table 4. Comparison of DNA electrophoresis in various gels.

Gel Baking Alkalin Salt (g/L) Water Other PH Comment
Soda e Buffer
(g/L) (g/L)
0.8% agar 0 0 0.05 Distilled -did not work
agar water -standard stayed in well
1.0% agar 0 0 0.05 Distilled -did not work
agar water -standard disappeared
0.8% agar 0 0 0.03 Distilled -did not work
agar water -not many bubbles present
during electrophoresis
-tracking dye and bands
disappeared
0.8% agar - - - - 1X TBE -good results with positive
agar control and samples
-bands were very tight
0.8% agar 0 0 0.10 Distilled -tracking dye turned yellow
agar water -bands not visible under
UV light
0.8% agar Yes No 0.05g/L Distilled 8.0 -tight bands
agar water
0.8% agar 1.71 0 0.05g/L Distilled 8.0 -tight bands, good results
agar water
0.8% agar 9.39 0 0.05 Distilled 8.3 -standard had a very faint
agar water band
-bands from samples were
not present
1.2% agar Yes 0 0.05 Distilled 6.0 -standard and sample bands
agar water were not visible
-tracking dye turned yellow
1.2% agar Yes 0 0.05 Distilled 7.0 -very faint bands present
agar water for standard and samples
1.2% agar Yes 0 0.05 Distilled 8.0 -faint bands present for
agar water standard and samples
1.2% agar Yes 0 0.05 Tap 7.0 -tracking dye turned yellow
agar Water -standard and samples’
bands were not visible
1.0% agar Yes 0 0.05 Distilled -added 8.0 -bands were not visible
agar water 10
drops
methyle
ne blue
to
buffer
-very
thin gel
0.8% agar Yes 0 0.05 Distilled -added 8.0 -bands were not visible
agar water 10
drops of
methyle
ne blue
to
buffer
-very
thin gel
1.0% agar Yes 0 0.05 Distilled -stained 8.0 -bands visible under UBC
agar water for 20 light and were very tight
min -staining did not work
then
destaine
d
1.0% agar - - - - 1X TBE - -excellent results
agar -tight bands were visible
under UV light from
positive control and
samples
1.0% agar Yes 0 0.05 Distilled -stained 8.0 -tight bands were visible
agar water for 2 under UV light from
hours positive control and
- samples
destaine -staining did not work; no
d for bands were visible
several
days
1% agarose - - - -1X TBE -excellent results
-tight bands were visible
under UV light from
positive control and
samples
1% agarose Yes 0 0.05 Distilled 8.0 -excellent results
Water -tight bands were visible
under UV light from
positive control and
samples
-stained bands were also
visible
1% agarose Yes 0 0.05

Staining Solution

To visualize the DNA bands in the gel, the gel was stained in a methylene blue solution.
Methylene blue consists of the salt methylene blue chloride. In water, the salt disassociates into a
positively charged methylene blue ion that is colored blue and a negatively charged chloride ion, which is
colorless. This blue chromophore is then able to bind to the positively charged DNA in the gel.
(Reference: [Link]
Methylene blue is a convenient stain to use in the lab because it is chemically safe, readily
available, reusable, and detects the presence of more than 20ng DNA/band. (Reference: Molecular
Research Cernter at wysiwyg://25/[Link]

Methylene blue can conveniently be found at most pet stores. It is generally sold as an aquarium
disinfectant at a 5% concentration. The best gel staining results were obtained using 0.02% methylene blue
in distilled water overnight at room temperature. Using this protocol, destaining with distilled water was
not required. Methylene blue staining solutions at higher concentrations were found to stain the gel too
dark, making it difficult to differentiate the background from the DNA bands even after destaining with
distilled water. Also, if the agar agar powder was not completely dissolved in the gel, the non-dissolved
flakes throughout the gel were stained dark blue, preventing the formation of distinct, visible bands.
Generally, it is recommended to not add methylene blue to the running buffer or gel since it provides no
clear advantage in the detection of the DNA bands. Attempts to incorporate methylene blue in the running
buffer at various concentrations were only successful once, but this was found to not be reproducible. If
methylene blue is added to the running buffer, it is recommended that the stain is completely homogenous
in the buffer, otherwise the gel will potentially be stained nonspecifically.

A stain of corn DNA in agar agar gels stained with methylene blue are shown in Figures 1 below.

Figure 1. Corn DNA stained

A comparison of the methylene blue stain versus the ethidium bromide stain is demonstrated in
Figures 2 and 3.
Figure 2 and 3: 1.0% Agarose gel stained with ethidium bromide (figure 2) and methylene blue (figure 3). Loaded 60µL of a 110µL
sample made of 100µL sample and 10 µL loading buffer. Lane 1 loaded with split pea DNA, lane 2 loaded white onion DNA, yellow
onion DNA, and corn DNA extracted using the basic buffer method. The gel was run at 81 volts for 45 minutes

Tracking dye
Dyes of various concentrations and composition were tested to determine its suitability as tracking
dye. After every run, each band was compared to the DNA loading buffer standard’s band. Two dyes
produced excellent results. In particular, Club House brand’s red and blue dyes produced very tight bands.
Club House blue dye ran closely behind the standard. However, Club House red dye ran much further than
the DNA loading buffer, possibly due to a smaller sized particle. The figure below shows the distance that
each tracking dye traveled. (Show figure)

The Club House blue tracking dye was prepared from 0.5mL glycerine, 0.1mL distilled water and
10 drops of dye. To ensure good results, wipe the outside of the pipette tip with a tissue before loading the
wells. This prevents the dye from nonspecifically staining parts of the gel.

We also tested other dyes. The green fabric dye was found to be not suitable because it ran
towards the negative electrode; it must, therefore, be positively charged. The hot blue fabric dye separated
into two bands, suggesting that this dye is made up of two differently sized molecules. In addition, the
bands smeared across the gel during the run. The cold blue dye was difficult to see, even at concentrations
higher than recommended. It was found to be a large molecule because it migrated the same distance as the
largest fragment in the lambda Hind 3 sample.

Attempts to find a tracking dye with different brands and colours were unsuccessful. These
included Club House brand yellow, green and blue food colouring. Strawberry Kool-Aid also didn’t work
when we used concentrations of 2mL glycerol, 2mL distilled water and 2 grams of Kool-Aid crystals.
Finally, food club dyes including red, yellow, green and blue food colouring were also unsuccessful.

Findings mentioned above are summarized in the Table 5 below.

Table 5. Summary of tracking dye composition and its effectiveness.

Brand Colour Composition Comments

Club House Red 2mL glycerol Did not work

2mL distilled water
25-30 drops food colouring
Club House Yellow 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Club House Green 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Club House Blue 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Kool-Aid Strawberry 2mL glycerol Did not work
flavour 2mL distilled water
25-30 drops food colouring
Food Club Red 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Food Club Yellow 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Food Club Green 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Food Club Blue 2mL glycerol Did not work
2mL distilled water
25-30 drops food colouring
Fabric Dye Hot Blue 0.5mL glycerol Did not work
0.1-0.5mL distilled water Separated into two streaks
0.03g dye
Club House Red 0.5mL glycerol Very tight bands
0.1-0.4mL distilled water Ran ahead of DNA
loading buffer
Club House Blue 1mL glycerol Very tight band
0.5mL distilled water Ran slightly behind DNA
15 drops dye loading buffer
Club House Blue Unknown concentration!? Very tight band
Ran almost right beside
DNA loading buffer
Fabric Dye Cold red 0.5mL glycerine Did not work
0.1mL distilled water

CONCLUSION