Alu Polymerase Chain Reaction Lab
April 2, 2015- April 3, 2015
Purpose: The purpose of this lab is to prepare a PCR, polymerase chain reaction, for an amplification of an Alu insert after isolating DNA from cheek cells.
Materials: Pipets(2-20; 20-200; 100-1,000 micro-liters), Pipet tips, Salt water(0.9 %), Micro centrifuge, Wast containers, Micro centrifuge tubes, PCR tubes, Agarose, TAE buffer(1x), Gel tanks, Gel molds, Loading dye, Tube racks, Primer mix, Master mix(DNA polymerase, reaction buffers), Distilled water, DNA(positive control), Sharpie, Chelex, Heat block, Thermal cycler
Explanations: Alu is a variance in a person's genes to cause a section of their DNA sequence to repeat. As prime mates have evolved, select individuals have slight alterations in their genes and a mass data collection is being formed to try to pinpoint the group of humans that are more likely to have the shift in DNA. The Alu gene does not affect any cognitive or physical actions. PCR stands for polymerase chain reaction which is the process of amplifying a specific set of DNA and creating thousands of the copied sequence. This method is essential in our lab because it will differentiate the number of base pairs more and the change of genes(if a person has the Alu gene or not) is easier to note. In this process, primers are added to create the origin of replication in the DNA so another strand of bases can match the original. In order to extract our cells, we swished a solution with nine percent salt because this is the same concentration of salt in our blood plasma. Chelex is a substance used to purify the DNA because there are certain elements that could possibly interfere during the PCR.
Procedure: In order to isolate the DNA from individual cheek cells, each student swished salt water in their mouth and spit into a cup to bring out cheek cells which contain DNA. To keep the results confidential, every picked a private number or PIN to use as a label instead of their name. One milliliter of the mixture was transferred into a micro centrifuge tube to spin in a micro centrifuge for one minute so the cell pallet to the bottom of the container. The excess supernatant, liquid on top of the pellet, was poured out before re-suspending the cells in approximately 100 micro liters of supernatant. This could be possible by flicking the bottom of the tube or racking the sample by dragging the container along the top of the tube racks. 50 micro liters of the suspended cells was added to a five percent container of Chelex, which is a purifying compound. After adding the Chelex and cells, the tube was heated at 99 degrees Celsius for ten minutes. While waiting, we labeled a sterile micro centrifuge tube with our PIN and 'DNA'. After releasing the pressure and spinning it again, 20 micro liters of the supernatant, not including any Chelex beads, was added to this tube. The isolated DNA samples were refrigerated until the PCR amplification was prepared.
Procedure: To perform the PCR amplification, we added 10 micro liters of the DNA sample, 20 micro liters of the Master mix, and 20 micro liters of the Primer mix to a tiny PCR tube. Throughout this process until the point where we placed the tubes into the thermal cycler, we kept the tubes and mixes on ice. The thermal cycler was set to 95 degrees for the first two minutes then executed thirty continuous cycles of 94 degrees(30 seconds) to 60 degrees(30 seconds) to 72 degrees(for two minutes). After two hours, the temperature of 72 degrees was held for ten minutes before stabilizing at 4 degrees*. For this portion of the lab, two positive and negative controls were tested. Instead of using the isolated DNA from cheek cells, selected students ran the same experiment with water for the negative baseline and Positive DNA for the positive control. After the DNA was properly cycled through heat ranges to promote the polymerase chain reaction, loading dye was added to the mix before running the samples on an agarose gel.
*All degrees are Celcius
*All degrees are Celcius
Electrophoresis Gel Calculations:
(40x)(V)=(1x)(500 mL) V=(500 mL)/(40) =12.5 mL The remaining volume of 487.5 mL was filled with distilled water. |
50 mL of 1x TAE mixed with 1 gram of agarose Heated mixture in microwave and swirled Repeat process until entirely dissolved |
Reflection and Class Results: On the far left is a DNA ladder starting at 1,000 base pairs separating at increments of 100. Unfortunately, my sample smeared across the entire gel so there are no exact bands to show if I have the Alu gene. Due to the extended amount of time our gels were in the stain(an entire weekend) most of the classes results were skewed and only eleven people had readable data. There were three possible outcomes: heterozygous, homozygous for the Alu gene, and homozygous for not having the Alu gene.