Name: withheld
2012-11-19
Biology/Mr.Mukwevho
Lab Report 4
Title: Analysis of DNA Using Restriction Enzyme and Gel Electrophoresis.
Purpose:
The purpose of this lab is to create genetic profile of given DNA fragment using
different kinds and combination of restriction enzymes and gel electrophoresis. By examining
the distance and number of fragments on the gel electrophoresis, the sources of DNA
fragments can be identified. The result will then be compared to the hypothesis.
Hypothesis:
It is not possible to assume the result until the experiment is actually done. The only
thing that can be assumed that, based on the number of DNA that exist in the DNA fragment,
the distance and the number of fragments on the electric field will be different or same. If the
DNA fragment is composed of different kinds of DNA, then depending on what kind of
enzyme it reacts with, the sample will travel different distance and leave different number of
fragments behind its track. However, if the DNA fragment is composed of one DNA, then
there will be no difference among the samples in terms of distance travelled and number of
fragment on the gel electrophoresis. Then based on the result of the experiment, it is possible
to find out if the DNA fragments came from same or different sources.
Discussion of Theory:
Gel electrophoresis is used to separate nucleic acids or proteins that differ in physical
properties, such as size, electrical charge, and etc. Gel electrophoresis separates DNA
fragments based on their rate of movement through agarose gel in an electric field. According
to the theory, the bigger or longer the fragment is, the less distance and slower it will travel
on the electric field. Because nucleic acid molecules carry negative charges, they all travel in
the same direction from negative pole to positive pole. As the DNA fragments travel, the
thicket of agarose gel fibers hinder longer molecules more than for shorter ones. As a result,
longer molecules are separated from the fragment and left behind on the track. This property
allows gel electrophoresis to separate molecules by their size and length. By this, gel
electrophoresis separates DNA molecules from DNA fragments into number of bands. And
the examination of pattern of bands can be used to identify different sources of DNA
fragment.
Restriction enzyme is what used to separate DNA fragment into smaller pieces.
Restriction enzymes digest DNA by cutting the molecule at specific locations called
restriction sites, creating blunt ends or sticky ends depending on the kind of restriction
enzyme used. One application of restriction enzyme is DNA mapping, the process of cutting
DNA at specific sequences with restriction enzymes. DNA mapping can be used to identify or
predict DNA from different sources by genetic signature that is unique to each organism.
In terms of analyzing the result, it is said that mathematical formulas have been
developed to describe the relationship between weight of DNA fragment and the speed it
travels. The base pair length (bp) is substituted for molecular weight of DNA fragment for
simplicity in determining the size of DNA fragment. However, since bps were not provided in
the experiment, the experiment will only consider the length DNA fragment travelled.
Because bps are not known, it is unnecessary to calculate speed of DNA fragments.
Materials:
1. Note to record data
2. 0.8% agarose solution
3. 1 X TAE buffer
4. Methylene blue stain
5. 20μL of DNA fragments prepared using restriction enzymes
6. Bulb transfer pipettes
7. Marker to mark the distance travelled
8. Gel electrophoresis chamber
9. Power supply
10. 20μL of DNA fragments prepared using restriction enzymes
11. Equipment to hold samples
12. Staining tray
Procedures:
1. Come up with a hypothesis or question.
2. Pour the liquid gel into the casting tray
3. Wait and do not touch until gel solidifies.
4. After the gel solidifies, remove the ends of casting tray. Place the tray in the
electrophoresis gel box making the wells head toward the negative end (black).
5. Fill the electrophoresis chamber with 1 X TAE buffer up to the level that is just
enough to cover the entire surface of gel.
6. Remove the comb that is used to make wells without ripping the wells. Wells should
be submerged under the 1 X TAE buffer as well.
7. Prepare DNA samples by first adding 5μL of each Lamda, PST I, EcoRI, HindIII,
restriction buffer, and restriction enzyme into Yellow, Violet, Green, Orange, Pink,
Blue tubes respectively.
8. Add 5μL of Lamda into all of the tubes except blue one.
9. Add 5μL of restriction buffer into all of the tubes except blue one.
10. Add 1μL of PST I into only violet tube.
11. Add 1μL of Hind III into only orange tube.
12. Add 1μL of EcoRI into only green tube.
13. Add 2μL of sample loading dye into yellow, violet, green, and orange tubes.
14. Centrifuge the tubes to make sure they are gathered at the center of tube.
15. Heat the tubes with hot water for 2~3 minutes or so. Then carefully remove tubes
from hot water without shaking them.
16. After preparing DNA samples, load gel with DNA samples using pipette or other
loading device. Make sure to use fresh loading device each time and record the order
of loaded samples.
17. Load DNA samples into a separate well in the gel
18. Slowly draw up the sample from the tube
19. Dip the pipette or loading device into well. Make sure not to rip the well.
20. Expel the DNA sample and draw the pipette or loading device out of well in the gel.
21. Discard the used pipette or loading device and use new one to load other DNA
samples.
22. Repeat the same procedure from 17~20 with other DNA samples, loading them into
different well in the gel.
23. Close the top of the electrophoresis chamber and connect it to the electrical power
supply, positive electrode to positive electrode, and negative electrode to negative
electrode – black to black and red to red.
24. Turn on the power supply and set the voltage.
25. Recommended to let DNA samples to electrophorese until the loading dye band is
about 1cm from the end of the gel. However in this experiment, due to limited time,
the loading dye bands reached only 1.2cm from its wells.
26. Turn off the power supply, disconnect the leads, and remove the lid of the
electrophoresis chamber.
27. Carefully remove the casting tray, making sure it won’t rip.
28. Slide the gel into a staining tray.
29. Measure the distance DNA samples traveled and record the data (in centimeters).
30. Organize and interpret data.
31. Compare with hypothesis and analyze result. Give explanation to errors if found.
Results/Data Collection/Analysis:
1.2cm
Restriction
Lamda
PST I
EcoRI
HindIII
Enzyme
Color of Tube
Restriction
Clear/Standard
Buffer
Yellow
Violet
Green
Orange
Pink
Blue
Distance
1.2cm
1.2cm
1.2cm
1.2cm
Not used
1.2cm
Traveled
It is shown that all of the DNA samples traveled the same distance. This shows that
all of the DNA samples came from the same source or organism. Using bp values for each
DNA samples the sizes of DNA fragments can be calculated. Then the graph showing the
relationship between molecular weight of DNA fragment and its speed can be found.
However, since the bp values of DNA samples are not known, the standard curve cannot be
found. Therefore, the fact that all DNA samples moved the same distance, meaning they all
came from the same source, is everything found from this experiment.
Conclusion:
According to the result, DNA samples are from the same source or organism because
all of them traveled the same distance and there were no band patterns. If each DNA sample
traveled different distance and had different band pattern, the conclusion would be that DNA
samples came from different sources or organisms. Although hypothesis did not predict
specific result, the result of this experiment follows the hypothesis, which stated that based on
the theory the DNA samples will either move different of the same distance. If the distance is
different than they came from different sources; if the distance is the same than they came
from the same source.
If the DNA samples actually came from different sources or organisms and the
experiment result was incorrect, then it is clear that something about the experiment was
incorrectly done. To list possibilities of the causes of error: incorrect amount of restriction
enzymes were added to tubes, broken well in gel, imperfect gel, time given for DNA samples
to travel, and other causes that are concerned with gel and restriction enzymes. Because those
factors are what determine the result of the experiment, any process incorrectly done will
bring incorrect result to the experiment.
Improvements recommended:
There is couple of ways to improve the results. One of them is to increase the time
given to DNA samples to travel along the gel. It is possible that the DNA samples didn’t have
enough time to travel, separate, and show band patterns. If more time were given, the result
might have been different.
Since the experiment processes require accuracy and delicacy, little mistakes can
greatly influence the overall result of the experiment. In order to prevent such mistakes, the
experimenter can either work their best not to make any mistakes or use devices that make it
easier for user to work on delicate operations with high accuracy.
To further the experiment, DNA samples can be more complicated. For instance,
DNA sample can be a piece of meat, milk, or animal skin. Such DNA samples are more
complicated than DNA samples used in this experiment. Furthermore, the result derived from
such DNA samples can be actually applied to real world, providing the composition of DNA
sample and its history. Doing so, the experimenter can identify different kinds of DNA
samples that came from different sources or organisms and relate them with history and
evolution of DNA.
CITATION
Reece, Jane B., Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky,
Robert B. Jackson, and Neil A. Campbell. "Ch. 20/Biotechnology." Campbell
Biology. 9th ed. San Francisco: Pearson Education, 2011. 396-422. Print.
"Investigation 9/Biotechnology: Restriction Enzyme Analysis of DNA." (R) Biology Lab
Manual for Students (2001). N.p.: College Board, 2001. 111-24. Print.
Reynolds, P.D. 1992. Mantle-mediated shell decollation increases posterior aperture size
in Dentalium rectius Carpenter 1864 (Scaphopoda: Dentaliida). Veliger 35:26-35.
Gapp, D.A., R.N. Taranto, E.F. Walsh, P.J. Favorito, and Y. Zhang. 1990.
Insulin cells are found in the main and accessory urinary bladders of the painted
turtle, Chrysemys picta. J. Exp. Zool. 254:332-337.
Stokes, D., L. Stokes, and E. Williams. 1991. The Butterfly Book. Little, Brown and Co., Boston.
96 pp.
Pearcy, R.W., and W.A. Pfitsch. 1994. Consequences of sunflecks for
photosynthesis and growth of forest understory plants. Pages 343-359 in E.D.
.