ALU-HUMAN DNA TYPING THROUGH PCR
This experiment is a qualitative experiment which shows if an individual has a specific dimorphic Alu element. We used a process called Polymerase Chain Reaction (PCR) to identify this Alu element. Introduction
Knowing whether or not an individual possesses a certain gene can be very important in scientific research. Do to this importance PCR allows scientist to locate these Alu’s relatively easy. Our variables in this experiment were the hairs of the test subject, the lysis solution, the time of the water baths, the time of vortexing, whether or not the reaction pellet dissolved, the microcentrifuge, the thermal cycler, the primer solution, the automatic cycler, the agarose gel, whether or not the electrophoresis apparatus was set up and used properly, the loading of the DNA samples in the wells of the gel bed, proper staining of the DNA, and finally the operator error. Our hypothesis is 50 % of the subjects DNA samples contain the genotype. Our null hypothesis is that the genotype is there by chance and there is no genotype in these samples. Experiment/ Methods
1. Obtain three to four hairs containing a sheath, a barrel-shaped structure (often white in color) encircling the shaft near the base of the hair. If necessary, sheaths can be cut from the remainder of the hair shaft. 2. Place the hairs in the bottom of a 1.5 ml screw-cap tube. 3. Obtain the lysis solution from your instructor.
4. Mix the lysis solution by vortexing or pipeting up and down. Before the chelating agent settles, quickly remove 150µl and add it to the tube containing the hair. 5. Make sure the hair sheaths are completely submerged in solution and are not stuck on the sides of the tube. 6. Place the tube in a 56°C water bath for 15 minutes.
7. Remove the tube from the water bath and allow it to cool for 30 seconds. 8. Vortex the tube for 15 seconds.
9. Check again that the hair sheaths are completely submerged in solution and are not stuck on the sides of the tube. 10. Place the tube in a float and place in boiling water for 10 minutes. 11. Remove the tube from water and cool on ice for 2 minutes. 12. Vortex the tube for 10 seconds.
13. Spin in a microcentrifuge for 30 seconds. Centrifuge the cell suspension carefully. 14. Carefully remove 50 µl of supernatant and transfer it to a clean 0.5 ml microcentrifuge tube. 15. Place the tube containing the supernatant on ice.
16. Transfer the PCR Reaction pellet™ with your initials. 17. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube. 18. Add the following to the pellet: PV92 primer solution 20µl, Cell DNA (supernatant) 5.0µl 19. Gently mix the PCR reaction tube and quickly spin it in a microcentrifuge to collect the entire sample at the bottom of the tube. Make sure the PCR reaction pellet™ is completely dissolved. 20. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. 21. Transfer the PCR Reaction pellet™ to the appropriate sized tube (e.g. 0.5 ml or 0.2ml) for your thermal cycler. 22. To prepare the PCR Control reaction, add the following to the pellet: PV92 primer solution 20.0µl, Control DNA 5.0 µl 23. Gently mix the control tube and quickly spin it in a microcentrifuge to collect the entire sample at the sample at the bottom of the tube. Make sure the PCR reaction pellet™ is completely dissolved. 24. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. 25. For automatic cycling, each student should place his/her PCR tube (and the optional control reaction) in the programmed thermal cycler. Follow the same cycling schedule if you are manually cycling in three water baths. Initial Denaturation 94°C for 5 min., 32 cycles @ 94°C for 30 sec., 61°C for 30 sec., 72°C for 45 sec., Final Extension 72°C for 4 min. 26. After the...
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