Gel Electrophoresis
Laura Gallagher Partner: Rob Einersen Biology Period D Mr. Alvarez 15 February 2013 Gel Electrophoresis Introduction: Agarose Gel Electrophoresis is a process in which the process of determining whether a strand of DNA is either positively or negatively charged. The container in which the gel is stored has a negative and positive side; whichever side the DNA molecules go to means the DNA is charged the opposite way. (Ware, Lunte, Gardiner)For example if a DNA molecule goes to the negative side that means that the DNA is positively charged and vice versa.
The agarose gel is composed of a fine powder substance, water and a buffer solution. The solution must be boiled to its boiling point then you have to pour the solution into a casting mold there also needs to be a comb that leaves six holds in the mold. It must be left in the casting mold for about a half an hour for the gel to solidify. Electrophoresis has a few different uses such as establishing the size of a strand or molecule of DNA or RNA. (Bowen) It can also be used to find out family members or criminals or tests of that manner. This is all possible because of the DNA’s charge and the charge of the molds.
Although if a DNA molecule is too big it is not going to be able travel fast through the so it is better for the experiment if the DNA molecules are small. To get the DNA small restriction enzymes are used. (Roberts) Restriction enzymes cut specific DNA molecules in half which helps with the travel through the gel. Before the electrophoresis machine was developed people used to use gravity to separate DNADNA molecules are going to be negative and smaller molecules are going to move farther than the larger molecules. The purpose of this lab is to learn how to create an agarose gel and properly load a well in an agarose gel.
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The purpose is also to learning how to use electrophoresis equipment and how to analyze the results of DNA electrophoresis. Methods and Materials: Start off, by gathering all of the necessary materials for the beginning which is 1. 2 mL of gel buffer in one graduated cylinder, in a separate graduated cylinder58. 8 mL of water and 0. 48 g of agarose powder. Next take an Erlenmeyer flask and mix all three of the substances, place the flask on a hot plate and keep swooshing the mixture in the flask. Repeat that step until the liquid in the hot plate becomes clear, make sure that the solution does not boil.
Let the solution sit until it is lukewarm. Now, the molding tray should be prepared by taping off the sides with painters tape so no liquid can escape. Place the comb in the designated slot in the molding tray and pour the agarose solution into the mold tray. Now, take a graduated cylinder and pour in 8 mL of the buffer solution and 400 mL of water, then, pour it into the gel box. After the gel solidifies, with extreme caution, remove the painters tape and comb. Next, take the mold tray and place it on the table; now, take six disposable pipettes and the colored dye from you teacher and fill each pipette with a different color.
After each pipette has a different color, empty the pipette not too far in or too close to the surface of each well. Next, place the mold tray in the gel box, close the lid, and plug it in for about a half an hour. After the half an hour is up, take a couple pictures of the mold tray and the results that you saw. Results: Figure 1 Figure 2 As you see in figure 1, the dye pigments have been placed in each well and it is not perfect some of the dye have gotten out and leaked out on the top of the gel.
That is okay though because the experiment was still a success because the dye pigments did move with the DNA molecules as you can see in figure 2. The dye pigments in all of the wells except for the well at the very top, all moved towards the positive side of the tray. The well at the very top contained positively charged DNA so that DNA started to move towards the negative side of the tray. Discussion: For the most part, my hypothesis was correct, I was correct about the smaller particles moving further than the larger particles. I was correct and incorrect when I said that The DNA molecules are going to be negative. I as correct because five out of six were negative, although there was one that was positive so it did not go in the same direction as the other five. DNA’s direction is only influenced by one factor, which is whether the DNA is negatively or positively charged. This directly affects why the DNA moved the positive pole because DNA is negative so due to attraction it goes to the positive side of the pole. When a molecule is moving its rate of length and speed correlates with its size, if a molecule of DNA is large, it is going to be harder for it to move so it would be much easier for a small molecule of DNA to move across the gel.
You can the molecules of DNA moving because they are dyed with the dye pigments. Electrophoresis causes the smaller DNA molecules to move further because it is easier for the positive charge to pull smaller pieces of DNA. Though, for this to happen, the power has to be turned on. Once the power is turned on it also turns on each end’s charge on each side of the tray so the DNA is attracted to that side of the tray. Most of the DNA the molecules carried a negative charge. A negative charge is carried because those molecules went towards the positive pole.
Although those few molecules carried a positive charge so they went to the negative pole. The banding patterns in the gel are determined by the size of the DNA molecule. It can be interpreted as some of the DNA molecules weren’t broken down as small as others. Scientists use the number of nucleotides in one sample and it is compared to another blood sample. All of the similarities and differences add up. Also the sequences of the bases in a chain of DNA. In many murder investigations, DNA is used to find the culprit. Such as the case of James Anagnos, James was beaten and stabbed to death in 1977 in his bar. NBC) James was holding a strand of hair that belonged to his murderer. Three decades later they compared the DNA is the strand of hair to a man named Frank Wright, it was a match. It didn’t serve any justice though, because Wright died in 2002. This is similar to the case of Priscilla Ann Blevins. Priscilla’s remains were found off an interstate and they were stored in a facility and kept under the name “Jane Doe”. (Lohr) Blevins’ genetic information was entered into a computer and it matched up with “Jane Doe” and was later confirmed with dental records.
DNA helped to resolve this 37 year old cold case just like it did for the 1993 murder of Alie Berrelez. Alie was a five year old girl who was sitting in her apartment complex eating pizza when she was kidnapped. (Curry) Alie’s remains were found four days later stashed next to a creek. Nick Stofer was the main suspect but they couldn’t arrest him because they didn’t have enough evidence against him. When this crime happened there was no such thing as DNA testing so there was no way they could prove it was him. In 2011, they compared DNA from Alie’s under to Nick Stofer and it was a match.
Again Stofer died before he could stand trial, he died in 2001. Bibliography B. R. Ware, Susan Lunte, Kathleen Gardiner, 2012, "Electrophoresis," in AccessScience, ©McGraw-Hill Education, Retrieved from http://www. accessscience. com/content. aspx? searchStr=Electrophoresis&id=226400 Bowen R. , 2000, “Agarose Gel Electrophoresis of DNA”, in Colostate, Retrieved from http://arbl. cvmbs. colostate. edu/hbooks/genetics/biotech/gels/agardna. html Richard Roberts, 2012, "Restriction enzyme,” in AccessScience, ©McGraw-Hill Education, Retrieved from http://www. accessscience. com/content. aspx? earchStr=restriction+enzymes&id=584150 2010, “Three Decade Old Murder Mystery Solved Using DNA” , in NBC California, retrieved from http://www. nbclosangeles. com/news/local/Three-Decade-Old-Murder-Mystery-Solved-Using-DNA-101944298. html Lohr D. , “After 37 Years, Priscilla Ann Blevins' Disappearance Solved Using DNA”, Huffington Post, Retrieved from http://www. huffingtonpost. com/2012/11/01/priscilla-ann-blevins_n_2059155. htmlCurry C. , 2011, “Cold Case of Murdered 5-Year-Old Alie Berrelez Solved”, ABC News, retrieved from http://abcnews. go. com/US/cold-case-year-murdered-1993-solved-dna/story? id=14510785
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