DNA Extraction and Agarose Gel Electrophoresis

Melan Anthony A. Yap 2011

Labmatb C36

October, 24,

DNA Extraction and Agarose Gel Electrophoresis I. Introduction ion  The main focus of the two experiments is the molecule of life or more scientifically known as deoxyribonucleic acid or DNA. The discovery of  DNA, its structure, and function was probably the most significant biological discovery of the 20th century. ce ntury. It has had a tremendous impact on the different fields of science. From identifying genes that lead to the development of diseases, to producing medicines and vaccines to treat them, identifying and analyzing genes has led to extraordinary breakthroughs that have changed the face of the future of science forever. As such, analysis of DNA is crucial in this modern time. Two of the analysis techniques that may be applied to DNA is DNA Extraction and Agarose Gel Electrophoresis. II. II. Obje Object ctiv ives es a. Extrac Extractt DNA from from green green peas peas b. Understand Understand the the principles principles and and process process of DNA extracti extraction on c. Know the the concepts concepts and importa importance nce of Agarose Agarose Gel Electro Electrophor phoresis esis d. Be familiar familiar with with the Agaros Agarose e Gel Electrop Electrophores horesis is Unit e. Understand Understand better better DNA through through photo-docume photo-documentati ntation on software software I. Ma Mate teri rial als s and and Met Metho hods ds Materials for DNA Extraction a. Gree een n pea peas s

b. Salt c. Cold Wa Water d. Liqu Liquid id Det Deter erge gent nt

e. f. g. h. i. j. k. l.

Pineapple Juice Ethyl Alcohol Blender Test Tubes Glass Stirrer Pipette Graduated Cylinder Beaker

Materials for Agarose Gel Electrophoresis a. Micropipettors b. Sterile White Tips

c. d. e. f. g. h. i.  j.

Petri Dish Parafilm Tissue Paper Latex Gloves Beaker 100 or 250 ml Flask Mupid Electrophoresis Unit Automatic Voltage Regulator k. Microwave Oven l. 0.5X TBE buffer m. Gel Loading Dye (Type III) n. Molecular Biology Grade Agarose o. DNA Samples p. 100bp DNA size/MW marker

Method for DNA Extraction

Method for Agarose Gel Electrophoresis I. Results or Data From the DNA Extraction activity, we are able collect 3 small test tubes containing DNA samples. The DNA samples looked like white cotton. From the Agarose Gel Electrophoresis on the other hand, the DNA “run” a little.  This signifies that the experiment was a success though it didn’t really “run” that much. II. Discussion DNA Extraction  The use of DNA for analysis or manipulation usually requires that it is isolated and purified to a certain extent. DNA is recovered from cells by the gentlest possible method of cell rupture to prevent the DNA from

fragmenting by mechanical shearing. A detergent is used to solubilize the cell membrane or cell walls. If physical disruption is necessary, it should be kept to a minimum, and should involve cutting or squashing of cells, rather than the use of shear forces. Purpose of the Different Materials Used: 

Salt- helps the DNA precipitate (solidify and appear) when alcohol is added.

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Cold Water- helps keep the DNA intact during the extraction process. Cooling slows down enzymatic reactions. This protects DNA from enzymes that can destroy it.

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Blender-breaks the cell wall of the plant

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Pineapple Juice- acts as the meat tenderizer -is a good substitute because the one of the most common enzymes used in meat tenderizer is Bromelain.  This enzyme is extracted from pineapple. The enzyme is a protease, meaning they break apart proteins.

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Alcohol- DNA precipitates in the presence of alcohol, which means it doesn't dissolve in alcohol. This causes the DNA to clump together when there is a lot of it.

 The white stringy/cotton-like stuff is actually a mixture of DNA and RNA.  The procedure for DNA extraction is really a procedure for nucleic acid extraction.

Agarose Gel Electrophoresis Agarose Gel Electrophoresis is done to check the purity and intactness of  the DNA extracted and also to determine the concentration of the DNA. Purpose of the Different Materials Used: 

Electrophoresis Buffer- usually Tris-acetate-EDTA (TAE) or Trisborate-EDTA (TBE).

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Loading buffer- contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded

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Ethidium bromide- a fluorescent dye used for staining nucleic acids.

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 Transilluminator- used to visualize ethidium bromide-stained DNA in gels.

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Agarose gel- provides a matrix with pores to allow molecules to travel through and be sorted by size

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Electric current- is the force that causes the negatively charged DNA molecules to more toward the positive pole

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Wells in the Gel- are the "starting gates" for the DNA molecules to be loaded into before starting the "race"

I. Conclusion and Recommendation

 The activity is important in our better understanding of DNA and of the course as a general. The activities were both educational and fun. It made us appreciate more the concepts and processes involved in biology or biotechnology. It was really a true embodiment of a transformative learning of the course. We learned biotechnology through experience and practical applications. The only recommendation that I might give is to allocate more time in the agarose gel electrophoresis.

II. References Molecular Biology and Biotechnology by J.M. Walker and R. Rapley Understanding Biotechnology by George Acquaah Biotechnology: A Guide to Genetic Engineering by Pamela Peters http://biology.arizona.edu/sciconn/lessons2/Alongi/Lesson5_3.htm http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.htm l http://learn.genetics.utah.edu/content/labs/extraction/howto/faq.html http://www.methodbook.net/dna/agarogel.html http://www.lifeindiscovery.com/dna/impact.html