Gladys Ericka Galang May 9, 2014

Rae Angelei Regalado May 9, 2014

Experiment No. 4 and 5
Isolation, Purification and Analysis of DNA Extracted from Duck Embryo
Abstract

DNA was extracted from a live duck embryo and the total weight extracted was 1.13 g with a percent yield of
48.09%. The concentration of DNA was calculated through the detection of the presence of nucleic acid using
double beam UV-vis spectrophotometry. The sample had a percent purity of about 10% with a concentration of
796.64 g/mL and the % m/v to be 11.3%. Agarose gel electrophoresis was done to further characterize the DNA
extract and to find out the molecular weight of the sample using the standard. The molecular weight of the sample
is found out to be 246.64 Da using the standard ProMega 1kb DNA ladder as a guide.

Introduction
DNA is holds the genetic code of organisms and is
passed from one generation to another. Complex as
it is, DNA has been studied for researches, advances
in medicine and technology (Voet, 2011) such as the
cloning of Dolly the sheep using successful somatic
cell nuclear transfer. These also include the rise of
genetically modified organisms through genetic
engineering. (Campbell, 2012) For all this process to
be conducted, DNA must be extracted and isolated
from a source.
DNA is difficult to extract in an intact and
undamaged form because of its large size and fragile
nature. There are many things to consider when
extracting DNA. These include the effect of pH,
temperature, ionic strength, cellular conditions, and
mechanical stress placed on the strands. (Boyer,
2000)
In the experiment, DNA is extracted and isolated
from a duck embryo. After a number of reactions,
DNA was precipitated through its reaction with
ethanol. DNA becomes insoluble in the addition of
an organic solvent that makes the solution less polar.
(Boyer, 2000)
The collected muscles from the live sample were
subjected into soft motion circular grinding because
rigorous grinding, shaking, stirring and other
methods may disrupt bonds and cause cleavage of
DNA chains. (Boyer, 2000)
Tris HCl buffer of pH=8 was used for the
suspension of the sample to extract the DNA from
the cell. The basicity of the buffer reduces
electrostatic interaction between DNA and histones
the proteins where DNA wraps itself around for
coiling and condensation during interphase
(www.unc.edu) and it also minimizes nuclease
activity and denatures other proteins. (Boyer, 2000)
SDS was added to act as denaturant of
deoxiribonucleases and other proteins that may
destroy the DNA. Chloroform denatures proteins
and is used in the experiment to further deproteinize
the solution. (Boyer, 2000)
Addition of NaCl in the solution was due to the fact
that DNA is most stable in salt solutions, even more
than it is in distilled water. (Boyer, 2000)
For the characterization of the DNA, double beam
UV-vis spectrophotometry was used in the
experiment. Because of the aromatic rings present in
the bases, changes in structure such as the
unwinding of the helix are detected because of its
effect on the absorption. Purity is also identified
through reading the absorbance of DNA at 260 and
280 nm where DNA and proteins, respectively, have
peak absorptions. The ratio A
260
/A
280
, as used in the
experiment, is used to measure the relative nucleic
acid/protein content of the DNA sample. (Boyer,
2000)
Another way to characterize the DNA is thermal
denaturation where the sample is treated with
denaturing agents and its absorption increases.
A
260(T)
/A
280(25C)
curve is plotted.
Another way is the binding and fluorescence of
Ethidium Bromide with the DNA which was done as
the second part of the experiment accompanied by
agarose gel electrophoresis. (Boyer, 2000)
Because of the size of the DNA which is larger
compared to a single protein analysed by
polyacrylamide gel electrophoresis, agarose gel
electrophoresis was used for the experiment.
Agarose is a linear polymer of galactopyranose
derivatives and is extracted from sea weed. Unlike
polyacrylamide, the gel is prepared horizontally
because it is fragile. (Boyer, 2000)
DNA is viewed under UV light after being soaked in
the Ethidium Bromide solution and undergoing
electrophoresis.
This experiment requires the estimation of the
concentration and purity and the characterization of
extracted DNA using spectrophotometric methods,
and agarose gel electrophoresis. The molecular
weight of the sample is to be calculated, as well.
Materials and Methods
The experiment was divided into two parts, the first
part being the extraction, purification and
quantification of DNA and the second part is the
analysis and preparation of the purified DNA
sample.
For the first part of the experiment, DNA was
extracted from a duck embryo. Meat weighing 2 g of
the sample was added with liquid nitrogen and was
homogenized through slicing and grinding.
The sample was then suspended in a 0.05 M Tris-
HCl buffer which was preheated at 55 C before
transferring the mixture to a conical tube. SDS was
added dropwise to the sides of the tube to get a final
concentration of 1% SDS respectively.
The solution was incubated in 55C water bath for
45 minutes, gently shaken every 10 minutes.
Addition of chloroform was done dropwise to the
sides of the tube. The solution was shaken and was
subjected to centrifugation twice for five minutes.
A wide-tipped Parteur pipette was used to collect the
aqueous layer which was then transferred to small
beaker where 5 M NaCl was added.
Ethanol was added to the sides of the beaker
resulting to a final 70% ethanol concentration. DNA
which appeared as the fibrous white precipitate was
spooled using a pre-weighed J-tube and was air-
dried. The J-tube with the DNA was weighed to
obtain the yield. Then the DNA was dissolved using
10 mL 0.05 M Tris-EDTA buffer. The concentration
and % (w,v) of the stock solution was obtained.
From the stock, 40 L of 10% (w,v) solution was
pipetted out and diluted to 5 mL using the Tris-
EDTA buffer. The rest of the solution was stored for
the second half of the experiment.
The absorbance of the solution was read at 260 and
280 nm against the Tris-EDTA buffer as blank.
From this, the ratio of A
260
and A
280
and DNA purity
was calculated from which the DNA was
concentration estimated.
Agarose gel electrophoreses was used to analyse the
stock solution in the second part of the experiment.
The gel was prepared from 0.25 g gel powder which
was mixed in 25 mL of 1X TAE buffer. The mixture
was homogenized through heating not boiling, with
occasional stirring. The transparent molten agarose
was allowed to cool to 37 C then was added with
300 L of ethidium bromide. The solution was
swirled to mix.
The solution was poured to the gel tray, avoiding air
bubbles into the mold as much as possible. The
comb was placed over but was not allowed to touch
the bottom of the gel. The gel solidified after 20-30
minutes at room temperature. The comb was
removed from the gel and the wells were flushed
with the buffer.
To two pieces parafilm set on the table, 30 L of the
loading buffer was added to which 20 L of the
DNA sample was also added. The top of the pipette
was used to mix the solution. From the resulting
solution, 20 L was loaded to the well.
The gel chamber was filled with running buffer until
the gel containing the sample was completely
immersed. At 100 V, the apparatus was ran for 30-
45 minutes until the length of the tracking dye
reached 80% of the gels.
The gel was removed from the setup and was placed
in a transparent flat-bottomed container. The gels
were placed on a UV light box.
Results and Discussions
From the original 2.35 g of the live organism, 1.13 g
of DNA was extracted, giving a percent yield of
48.09%. After dissolving DNA in 10 mL Tris-
EDTA buffer, the concentration was calculated to be
11.3% (w/v).
Under spectroscopic analysis, the ratio of A
260
/A
280

was calculated to be 1.244 and using the table
below, the purity can be estimated to be about 10%.
Table 1. Nucleic Acid Correlation Factor
A
260
/A
280
% nucleic acid
0.57 0
1.06 5
1.32 10
1.48 15
1.59 20
1.67 25
1.73 30
1.78 35
1.81 40
1.84 45
1.87 50
1.89 55
1.91 60
1.93 65
1.94 70
1.95 75
1.97 80
1.98 90
1.99 95
2.00 100

Assuming 50 g/mL corresponds to A
260
/A
280
= 1,
the DNA concentration can be estimated to be
796.64 g/mL and the % m/v to be 11.3%. The low
percent concentration can be indicative of the low
purity of the DNA sample, and the presence of
possible RNA contaminants in the solution.
The figure below shows the DNA bands viewed
under UV light.

Figure 1. DNA bound to EtBr under UV light
The molecular weight of the DNA can be calculated
using the distance travelled of the stain which is
equal to the log molecular weight of the standard
used. The relative mobility of the DNA, or its R
f

value was also calculated from the standard. Using
the guide standard of ProMega 1kb DNA ladder
(Ref# G571A) of 1% agarose as used in the
experiment, we get the calibration curve.


Figure 2. ProMega 1kb DNA ladder of 1% Agarose
gel (Ref# G571A)

The R
f
values of the standard are tabulated and
graphed as follows.

Table 2. R
f
values of the standard
BP
Dye front
distance
Band
distance
Rf
250 34 12 0.353
500 34 17 0.500
750 34 23 0.676
1000 34 26 0.765

Figure 3. Graph of Log MW vs. Rf values


The calibration curve of the graph has a line
equation of:
y = 1.4038x + 1.9379

We can input the R
f
values of the wells from the
data drawn from the experiment in place of x,
particularly from the 3rd and 7th well to get the y
value which is the molecular weight of the DNA
sample. Since the band on the 3rd well is not
prominent, the data from the 7th band is taken into
consideration. The R
f
value of the 7th band is 0.324
and therefore molecular weight from the equation is
246.64 Da.

Conclusion
Spectrophotometric analysis is a relatively good way
to analyse the data given besides the error of
turbidity of the sample. Agarose gel electrophoresis
proved to be an effective way to calculate for the
molecular weight of the sample although there are
problems encountered with the
Possible sources of errors in DNA extraction include
improper handling of reagents, and unwanted
cleavage of DNA fibers. Nucleases that may not
have been denatured might have caused nucleic acid
degradation and therefore a lower yield. Also,
inefficient spooling of DNA could have decreased
the yield.
Recommendations for further experimentations
would be the use of sample with higher nuclear
cytoplasmic ratio chicken liver, calf thymus, white
blood cells, saliva, hair follicle, and bone. The
electrophoresis set up could use fresh extracts of
DNA for better binding with EtBr solution. The use
of 0.3 to 2.0% agarose gel may also be
recommended as they are most effective in nucleic
acid separation. Further study about the structure of
the DNA extracted may also be added to the
experiment. A separate experiment to test the for the
conformation of the DNA sample may also be
conducted using different concentrations of ethidium
bromide. (Boyer, 2000)

References
Boyer, R., Modern Experimental Biochemistry, San
Francisco, California: Benjamin/ Cummings. 2000
Campbell, Biochemistry. Brooks/Cole, Cengage
Learning, 2012
Voet, D. et al., 2011, Biochemistry,
Courier/Kendallville
Histones What are those?
<http://www.unc.edu/depts/our/hhmi/hhmi-
ft_learning_modules/2011/proteinsmodule/histones/i
ndex.html>
ProMega 1kb DNA ladder (Ref# G571A)
<http://www.unifr.ch/biol/ecology/lexer/Reagents/1
kb%20DNA%20Ladder%202.pdf>
y = 1.4038x + 1.9379
R = 0.9753
2
2.2
2.4
2.6
2.8
3
3.2
0.200 0.400 0.600 0.800