Amit Kumar Nick Sigismondi Joseph Seares 19 February 2014 A.P.

Biology Lab Investigation 9 Biotechnology: Restriction Enzyme Analysis of DNA Activity III: The Basics of Gel Electrophoresis

Purpose: After DNA cloning produces large quantities of specific DNA segments, the process of Gel Electrophoresis can help answer a myriad of inquiries such as where in the body and when certain genes are expressed, whether a certain gene differs from person to person, and even what role genes play in an organism. This technique utilizes a gel made of a polymer (for example, the polysaccharide agarose), which acts as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge, and other physical properties. In modern society, biotechnology is relevant, as it extends beyond what we see on television crime shows. Apart from determine what ingredients are in the meat of a hamburger, DNA technology can be employed to determine paternity, diagnose an inherited illness, and even solve historical mysteries. Overall, the purpose of conducting this experiment is to ascertain a better grasp of Gel Electrophoresis and its applications to our daily lives. Materials: 20 microliter vials of DNA Fragments prepared using restriction enzymes Rack for holding samples

3 plastic bulb transfer pipettes (or similar devices) Permanent Marker Gel Electrophoresis Chamber Power Supply Staining Tray Ruler 0.8% agarose solution 1 X TAE (tis-acetate-EDTA) buffer Methylene Blue Stain Writing Utensil Paper to Record Data

Procedure: Part I. Casting the Agarose Gel 1. Seal the ends of the gel-casting tray with tape or something similar. 2. Insert the well-forming comb. This will produce the areas where the DNA will enter the Gel Electrophoresis system. 3. Carefully pour agarose gel solution into the gel-casting tray to a depth of about 5 to 6 mm. This depth should cover roughly one half the height of the comb teeth. a. While still liquid, use a pipette to remove any bubbles in the agarose solution if necessary. 4. Wait 15 to 20 minutes until the gel solidifies. In doing so, it will become cloudy. Do not disturb or touch the gel while it is solidifying.

5. When the gel has finally settled after 15 to 20 minutes, remove the ends of the gel-casting tray. The gel, now solidified, should not seep out of the tray. 6. Place the gel-casting tray into the electrophoresis gel box so that the comb is at the negative (black) end. 7. Fill the box with 1 X TAE buffer, to a level that just covers the entire surface of the gel. 8. Remove the comb from the system. Be sure that the wells left by the comb are completely submerged in the buffer solution. Upon removal, there should be seven wells created. a. This is how the finished system should look thus far:

Part II. Loading the Gel 9. Load 15 20 microliters of each sample of DNA into a separate well in the gel. a. Using a pipette, carefully draw out some of the DNA solution from the test tube it is packaged in. b. With steady hands, release the pipette into the desired well. i. NOTE: Expel any air in the end of the pipette before loading the DNA sample. 10. Repeat Step 9 for the rest of the DNA samples. Make sure to use fresh pipettes for each DNA sample. Part III. Electrophoresis 11. Close the top of the electrophoresis chamber and then connect the electrical leads to an appropriate power supply, positive electrode to positive electrode, and negative electrode to negative electrode. 12. Turn on the power supply and set the voltage as directed by the instructor. 13. Observe the movement of DNA from the wells to the opposite side of the system. 14. Once the DNA has completed its movement, turn off the power source, remove the lid of the electrophoresis chamber, and record the data.

Data: Figure 1: Measured DNA Diagram Electrode ()

Electrode (+)

Photograph of Figure 1

Conclusion: As evidenced by this lab, Gel Electrophoresis is a crucial component of the upand-coming field of Biotechnology, as it holds a very lofty place in DNA sequencing. Indeed, techniques such as Southern, Northern, and Western blotting would not exist if not for Gel Electrophoresis. This excellent procedure, used to systematically organizes nucleic acids or proteins on the basis of size, electrical charge, and other physical properties. The method is also crucial in crime investigations, which suspects DNA are compared to the DNA left behind at the scene of the crime. This lab explored this particular application of Gel Electrophoresis primarily, with the utilization of a story within the plot. In this plot, several students within A.P. Biology find their laboratory in chaos and their teacher missing. Instantly, the two students, Marcus and Laurel, go about attempting to solve this perplexing mystery. The process of Gel Electrophoresis begins with restriction enzymes that cut DNA strands at particular points, creating thousands of restriction fragments of varying size and length. Gel Electrophoresis can then be employed to sequence this plethora of DNA fragments, as it can organize it in terms of several possible physical properties. As was done in the lab, each one of the suspects DNA as well as the DNA found at the scene of the crime would be injected in the Gel Electrophoresis system. In this lab, however, an additional DNA sample was added: DNA Marker. Stated simply, this DNA marks consists of a range of DNA fragments of known size and number of base pairs, and therefore helps calculate the size of the samples being experimented on. Additionally, note that in the system the wells are always placed near the negative side of the power source. This is because nucleic acid molecules carry negative charges on their

phosphate groups, and thus travel toward the positive pole on the opposite side of the Gel Electrophoresis system (Procedure, Questions 1, 2, and 3). Gel Electrophoresis utilizes this to sequence the fragments, as longer DNA fragments tend to slow quicker than small DNA fragments, and thus are found nearer to the wells. In essence, the closer a DNA fragment is to a well, the longer it is. At the culmination of the experiment, it was observed that the results yielded from the DNA of Suspect # 2 was more closely related to the DNA of the crime scene than that of Suspect # 1. This conclusion, however, has a huge possibility of being erroneous, for there were a myriad of mistakes done in this experiment. First, in the experiment, the lab group received the materials last, and thus were left with a lesser amount of the DNA samples. In addition, when pouring the final dregs of the liquid agarose solution, a partially solidified chunk of agarose fell into the system, throwing it off tremendously, as the instructor even had to heat the system to liquefy the chunk. These are two of the main reasons for why much of the markers at the culmination of the DNA fragments movement were impossible to detect. In addition to this, the distances could have been measured wrong. Thus, the combination of the circumstances involved in the experiment as well as human error both establish that the conclusion drawn in this experiment has a large margin of error, and thus could yield incorrect data, possibly leading to the arrest of an innocent person.