Alu TestIn this lab we extracted our DNA so we could identify who had a chromosome called Alu. In our group there was Marsalina, Grace, Shasta., Sasha, Patrick, and Noah. The way this happens is through P.C.R.. P.C.R. or Polymerase Chain Reaction is when DNA is separated so we can see if there is Alu in it.
Purpose: The purpose this lab was to find out if we had alu in our chromosomes or not. By determining this we found out if we were homozygous or heterozygous.
Hypothesis: I presume that i will have one of the 3 geno types. Also that the whole class will be diverse in geno types.
Materials:
.9% of saline solution student cheek cells 5% chelex primer mix master mix TBE buffer Agarose gel, tracking dye DNA stain-"gel red" |
Procedure:
1. Put on gloves and obtain one 1.5 ml microcentrifuge tube containing 10% Chelex and use a permanent marker to label the top with an assigned DNA sample number. (Note that Chelex is not actually a solution, but a slurry of resin coated beads in water. You will see the beads settled at the bottom of the tube.)
2. Also obtain one 15-ml centrifuge tube containing 10 ml of sterile saline (0.9% NaCl) and label the side of the centrifuge tube with the same assigned DNA sample number.
3. Pour the contents (saline) of the conical centrifuge into your mouth and agitate vigorously for 2 min, using your teeth to gently scrape the inner surface of your mouth to dislodge as many cells as possible. The more cells in the sample, the higher the yield of DNA will be. [Note: this works best if you have not eaten/chewed gum for an hour. If you just ate something sticky like Doritos, you should try to rinse you mouth a bit first with water, because otherwise you end up with lots of food particles in the cell suspension.]
4. Expel (i.e., spit) the solution carefully into a clean 50 ml centrifuge tube, then carefully pour the saline/cheek cell suspension back into the original 15 ml centrifuge tube.
5. Place the 15-ml tube in the clinical centrifuge, putting your tube across the rotor from another for balance. Spin the tubes in the centrifuge for 5 min at 3,000 rpm. While the cells are spinning, eat some cookies in the lobby to get the salt taste out of your mouth.
6. When the spin ends, gently pour off the supernatant into the sink, leaving behind the pellet of cheek cells (and whatever else). You should see a white pellet of cells in the tube.
7. Use a sterile, graduated transfer pipette to draw the water away from the settled Chelex beads in the microcentrifuge tube. Use this water and pipette to gently resuspend the cells in the 15-ml tube. Then, transfer the cell suspension back to the microcentrifuge tube with the Chelex. Mix the cell/Chelex suspension well.
8. Firmly shut the microcentrifuge tube and place it in a floating rack with in the boiling water bath for 10 min. DO NOT DELAY BETWEEN STEP 7 AND 8.
9. When the 10 min boiling step has ended, use forceps to remove the tube from the water bath, and place your boiled sample on ice for 1-2 minutes.
10. Spin tube with others (making sure the rotor is balanced) for 5 min in the high-speed microcentrifuge. This will pellet the Chelex beads and cell debris, leaving the DNA suspended in the supernatant above the beads. IF YOU HAVE TO WAIT FOR OTHERS TO SPIN, KEEP YOUR TUBE ON ICE WHILE WAITING.
11. Wearing gloves, use a P200 micropipettor to transfer ONLY 150 µl of the clear supernatant from the top of the solution to a new, sterile microcentrifuge tube. Be careful not to transfer any of the material in the pellet because this will inhibit the PCR reaction.
12. Label the new tube with its respective DNA sample number. You have now isolated a small quantity of human genomic DNA. Keep the tube on ice until you are ready to set up your PCR reaction. The sample can also be frozen for later use. 4 B. PCR Amplification
1. Put on gloves. Use a permanent marker to label the top of a sterile 0.2 ml thin-walled microcentrifuge tube with your DNA sample number (as #2 above). This is your PCR reaction tube.
2. In the ice bucket on your bench you will find a tube labeled Amp Mix. Use a P-200 micropipettor to pipet 30 µl of this amplification mixture into your 0.2 ml PCR reaction tube. This mixture contains oligonucleotide primers, the four deoxynucleotides (A, T, C, G), Taq polymerase, buffers and tracking dyes for gel electrophoresis.
3. Using a P-20 micropipettor, pipet 10 µl of your extracted DNA sample into the labeled PCR tube. Place the 1.5 ml tube with the rest of your DNA solution on ice.
4. Mix the solutions and spin down your sample in the microfuge.
5. Your DNA is now ready for amplification in the PCR machine, or thermal cycler. Your instructor will collect your reaction tube, place it in the PCR machine, and start the program.
6. One person should also prepare a negative control (10 µl of water + 30 µl of Amp mix) and a positive control (10 µl of DNA solution from a known heterozygous individual + 30 µl of Amp mix) for the group.
7. The reaction will proceed as follows: 1 cycle 95˚C for 3 minutes (initial denature) 30 cycles 95˚C for 30 seconds (denature) 58˚C for 40 seconds (anneal) 72˚C for 90 seconds (extend) 1 cycle 72˚C for 10 minutes (final extension)
Day 2: C. Gel Electrophoresis of PCR Product
1. Your instructor will return your PCR reaction tube to you. Your reaction tube now contains your PCR product.
2. Centrifuge the tube for 10 seconds before opening the lid.
3. With assistance from your instructor, use a P-200 micropipettor to load the entire 40 µl into one well of a 2% agarose gel. Record your DNA sample number on the gel diagram sheet next to the appropriate well number for your PCR product. This is so you will know which lane of the gel contains your PCR product.
4. One person should load 10 µl of the 100-bp DNA ladder in Orange G into one well of each row of the gel.
5. The gel will run for ~1 hour at 100V. You will examine the stained gel this afternoon.
1. Put on gloves and obtain one 1.5 ml microcentrifuge tube containing 10% Chelex and use a permanent marker to label the top with an assigned DNA sample number. (Note that Chelex is not actually a solution, but a slurry of resin coated beads in water. You will see the beads settled at the bottom of the tube.)
2. Also obtain one 15-ml centrifuge tube containing 10 ml of sterile saline (0.9% NaCl) and label the side of the centrifuge tube with the same assigned DNA sample number.
3. Pour the contents (saline) of the conical centrifuge into your mouth and agitate vigorously for 2 min, using your teeth to gently scrape the inner surface of your mouth to dislodge as many cells as possible. The more cells in the sample, the higher the yield of DNA will be. [Note: this works best if you have not eaten/chewed gum for an hour. If you just ate something sticky like Doritos, you should try to rinse you mouth a bit first with water, because otherwise you end up with lots of food particles in the cell suspension.]
4. Expel (i.e., spit) the solution carefully into a clean 50 ml centrifuge tube, then carefully pour the saline/cheek cell suspension back into the original 15 ml centrifuge tube.
5. Place the 15-ml tube in the clinical centrifuge, putting your tube across the rotor from another for balance. Spin the tubes in the centrifuge for 5 min at 3,000 rpm. While the cells are spinning, eat some cookies in the lobby to get the salt taste out of your mouth.
6. When the spin ends, gently pour off the supernatant into the sink, leaving behind the pellet of cheek cells (and whatever else). You should see a white pellet of cells in the tube.
7. Use a sterile, graduated transfer pipette to draw the water away from the settled Chelex beads in the microcentrifuge tube. Use this water and pipette to gently resuspend the cells in the 15-ml tube. Then, transfer the cell suspension back to the microcentrifuge tube with the Chelex. Mix the cell/Chelex suspension well.
8. Firmly shut the microcentrifuge tube and place it in a floating rack with in the boiling water bath for 10 min. DO NOT DELAY BETWEEN STEP 7 AND 8.
9. When the 10 min boiling step has ended, use forceps to remove the tube from the water bath, and place your boiled sample on ice for 1-2 minutes.
10. Spin tube with others (making sure the rotor is balanced) for 5 min in the high-speed microcentrifuge. This will pellet the Chelex beads and cell debris, leaving the DNA suspended in the supernatant above the beads. IF YOU HAVE TO WAIT FOR OTHERS TO SPIN, KEEP YOUR TUBE ON ICE WHILE WAITING.
11. Wearing gloves, use a P200 micropipettor to transfer ONLY 150 µl of the clear supernatant from the top of the solution to a new, sterile microcentrifuge tube. Be careful not to transfer any of the material in the pellet because this will inhibit the PCR reaction.
12. Label the new tube with its respective DNA sample number. You have now isolated a small quantity of human genomic DNA. Keep the tube on ice until you are ready to set up your PCR reaction. The sample can also be frozen for later use. 4 B. PCR Amplification
1. Put on gloves. Use a permanent marker to label the top of a sterile 0.2 ml thin-walled microcentrifuge tube with your DNA sample number (as #2 above). This is your PCR reaction tube.
2. In the ice bucket on your bench you will find a tube labeled Amp Mix. Use a P-200 micropipettor to pipet 30 µl of this amplification mixture into your 0.2 ml PCR reaction tube. This mixture contains oligonucleotide primers, the four deoxynucleotides (A, T, C, G), Taq polymerase, buffers and tracking dyes for gel electrophoresis.
3. Using a P-20 micropipettor, pipet 10 µl of your extracted DNA sample into the labeled PCR tube. Place the 1.5 ml tube with the rest of your DNA solution on ice.
4. Mix the solutions and spin down your sample in the microfuge.
5. Your DNA is now ready for amplification in the PCR machine, or thermal cycler. Your instructor will collect your reaction tube, place it in the PCR machine, and start the program.
6. One person should also prepare a negative control (10 µl of water + 30 µl of Amp mix) and a positive control (10 µl of DNA solution from a known heterozygous individual + 30 µl of Amp mix) for the group.
7. The reaction will proceed as follows: 1 cycle 95˚C for 3 minutes (initial denature) 30 cycles 95˚C for 30 seconds (denature) 58˚C for 40 seconds (anneal) 72˚C for 90 seconds (extend) 1 cycle 72˚C for 10 minutes (final extension)
Day 2: C. Gel Electrophoresis of PCR Product
1. Your instructor will return your PCR reaction tube to you. Your reaction tube now contains your PCR product.
2. Centrifuge the tube for 10 seconds before opening the lid.
3. With assistance from your instructor, use a P-200 micropipettor to load the entire 40 µl into one well of a 2% agarose gel. Record your DNA sample number on the gel diagram sheet next to the appropriate well number for your PCR product. This is so you will know which lane of the gel contains your PCR product.
4. One person should load 10 µl of the 100-bp DNA ladder in Orange G into one well of each row of the gel.
5. The gel will run for ~1 hour at 100V. You will examine the stained gel this afternoon.
Results: For my result i ended up being -,- meaning i was homozygous.
Analysis: By being homozygous this means that i have a different ancestor than someone who is heterozygous. And since i am -,- i have no alu in my body
Conclusion: In this test i hypothesized that i would have one of the geno types. I was correct about this and was correct that the class would be diverse on it too. Some problems we had was our first experiment didn't work so we had to redo it. Other then that it was smooth sailing.
Analysis: By being homozygous this means that i have a different ancestor than someone who is heterozygous. And since i am -,- i have no alu in my body
Conclusion: In this test i hypothesized that i would have one of the geno types. I was correct about this and was correct that the class would be diverse on it too. Some problems we had was our first experiment didn't work so we had to redo it. Other then that it was smooth sailing.