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DNA Restriction and Electrophoresis Agarose gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. The main purpose of this technique is to separate or fractionate molecules apart from one another. This technique involves moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate further than longer ones Background: Agarose, which is extracted from seaweed, is a linear polysaccharide consisting of the repeating modified galactose units. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab containing a matrix that acts like a molecular sieve. During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical current. Under an electrical field, DNA will move to the positive electrode and away from the negative electrode. Several factors influence how fast the DNA moves, including; the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA. Restriction Enzymes Bacteriophages are viruses that infect bacteria by injecting their own DNA into the bacterial cell and force the bacteria to multiply the viral DNA. Bacteria have responded by evolving a natural defense, called restriction enzymes, to cut up and destroy the invading DNA. These enzymes search the viral DNA for specific palindromic sequences of base pairs, such as (GAATTCs), and cut up the DNA into pieces at these sites. The actual place in the palindrome where the DNA is cut is called a restriction site. An important feature of restriction enzymes is that each enzyme only recognizes a specific palindrome and cuts the DNA only at that specific sequence of bases. A palindrome can be repeated a number of times on a strand of DNA, and the specific restriction enzymes will cut all those palindromes at their restriction sites. Procedure Day One: Restriction Digest Preparation: 1. Label four 1.5mL microcentrifuge tubes as follows: B = BamHI digest E = EcoRI digest H = HindIII digest C = Control 2. Pipet the reagents into each tube according to the table below (Use a fresh tip for each transfer): Tube DNA B 4l E 4l H 4l C 4l Buffer BamHI EcoRI HindIII 5l 1l 5l 1l 5l 1l 5l Water 1l 3. Mix the components by gently flicking the tube with your finger and tapping gently on the table to collect liquid to the tube bottom. Pulse-spin in a microcentrifuge to collect all the liquid to the bottom of the tube. 4. Incubate the tubes for 1 hour at 37°C. Agarose Gel Preparation (0.8% agarose gel): 1. Weigh out 0.8g of agarose. 2. Add 100ml 1X TBE Buffer and 20l SYBR Safe stain to the agarose. 3. Using the hot plate and a stir bar, bring the agarose solution to a boil. *Be careful to NOT allow the solution to boil over! 4. Prepare gel cast and well comb. 5. Once agarose solution has cooled a bit, pour the solution into the gel cast. Allow the get to solidify. 6. Once your gel has solidified label it, wrap it in Saran wrap and place it into the refrigerator until the next lab period. Procedure Day Two: Prepare restriction digests for loading and sample loading: 1. Remove restriction digests from the freezer and allow them to thaw. 2. Remove your gel from the refrigerator, remove the Saran wrap and allow the gel to warm to room temperature. 3. Once the digests have thawed, pulse-spin them in a microcentrifuge to collect all the liquid to the bottom of the tube. 4. Add 3l of loading dye to each reaction tube. 5. Label a separate microcentrifuge tube “SM” (for size marker). To this tube add 10l HindIII, 10l dH2O and 3l loading dye. 6. Gently remove the comb, pulling it straight up and out of the set agarose. Place the gel along with the casting tray in the electrophoresis chamber that the wells made by the comb are at the negative electrode. 7. Fill the electrophoresis apparatus with 1X TBE buffer so that the entire gel is submerged. 8. Load the entire contents of each of your reactions into a separate well. Be sure to note the order in which you loaded your samples. 9. Close the top of the electrophoresis apparatus, connect the electrical leads to the power supply. 10. Turn on the power supply and electrophorese for approximately 45 minutes at 150V. 11. Turn off the power supply, disconnect leads from inputs and remove the top of the electrophoresis box. 12. Visualize and photograph your gel.