purpose
To determine the frequency of the Alu insert in the class using PCR.
Hypothesis
I think that I am a homozygeous.
Materials
Cheek Cell
Chelex
Micropipets
Micropipet Tips
Sterile Tubes
Plastic Beaker
Gel Box
Master Mix (nucleotides, DNA polymerase)
Tube rack
Reaction Tubes
Permanent Markers
Prime Mix
TBE: Buffer, Agorose Gel
DNA Stain Solution
Centrifuge
Chelex
Micropipets
Micropipet Tips
Sterile Tubes
Plastic Beaker
Gel Box
Master Mix (nucleotides, DNA polymerase)
Tube rack
Reaction Tubes
Permanent Markers
Prime Mix
TBE: Buffer, Agorose Gel
DNA Stain Solution
Centrifuge
Procedure
1. Swirl 10ml of saline solution in your mouth for 30 seconds and spit it into a cup to mix the cells.
2. Transfer 1000ul to 1500ul of the saline in your labelled tube
3. Spin your saline cell suspension in a microcentrifuge for a minute.
4.Make sure there is 100ul of saline remaining in the tube, flick or rack the tube to mix, resuspending the cell
5. Label a tube of chelex and add 50 ul of your cell suspension into the tube.
6. Take your tube to a heat block station and leave it there for 10 minutes.
7. After heating, remove the cap lock to release pressure, then get another clean microfuge tube and label it, write DNA on this tube
8. Use a P-200 to withdraw 50ul of supernatant from the chelex/DNA tube to your new tube. Make sure that you do not transfer any chelex beads.
9. Put your DNA tube in the class rack. Your teacher will refrigerate it until you are ready to prepare your PCR amplification.
10. Take a small PCR tube and pipet 20 ul of Master mix into PCR tube
11. Change your pipet tip and add 20ul of Primer Mix into your PCR tube
12. Add 10ul of your extracted DNA into the PCR tube
13. Place your reaction into the thermal cylinder
14. Get your PCR tube and place it in a balanced configuration in a microcentrifuge. Spin it for 10 seconds to bring the liquid to the bottom of the reaction tube.
15. Add 5ul of loading dye to your PCR tube
16. Carefully load 15 to 20 ul of the DNA/loading dye mixture into a well in your gel.
17. One student should load 5-10ul of 100 bp ladder into one of the wells of each gel.
18. When all of the samples are loaded, attach the electrodes from the gel box to the power supply, then electrophorese your samples at 150 volts for 25-40 minutes.
19. After electrophoreses, stain the gel and take a picture
2. Transfer 1000ul to 1500ul of the saline in your labelled tube
3. Spin your saline cell suspension in a microcentrifuge for a minute.
4.Make sure there is 100ul of saline remaining in the tube, flick or rack the tube to mix, resuspending the cell
5. Label a tube of chelex and add 50 ul of your cell suspension into the tube.
6. Take your tube to a heat block station and leave it there for 10 minutes.
7. After heating, remove the cap lock to release pressure, then get another clean microfuge tube and label it, write DNA on this tube
8. Use a P-200 to withdraw 50ul of supernatant from the chelex/DNA tube to your new tube. Make sure that you do not transfer any chelex beads.
9. Put your DNA tube in the class rack. Your teacher will refrigerate it until you are ready to prepare your PCR amplification.
10. Take a small PCR tube and pipet 20 ul of Master mix into PCR tube
11. Change your pipet tip and add 20ul of Primer Mix into your PCR tube
12. Add 10ul of your extracted DNA into the PCR tube
13. Place your reaction into the thermal cylinder
14. Get your PCR tube and place it in a balanced configuration in a microcentrifuge. Spin it for 10 seconds to bring the liquid to the bottom of the reaction tube.
15. Add 5ul of loading dye to your PCR tube
16. Carefully load 15 to 20 ul of the DNA/loading dye mixture into a well in your gel.
17. One student should load 5-10ul of 100 bp ladder into one of the wells of each gel.
18. When all of the samples are loaded, attach the electrodes from the gel box to the power supply, then electrophorese your samples at 150 volts for 25-40 minutes.
19. After electrophoreses, stain the gel and take a picture
result
For the result I found out that I was actually homozygous like I expected.
Analysis
We had to try three times taking longer than expected to finish this lab. I learned lots of things, mostly to follow instructions slowly and thoroughly. We had to restart many times due to not following instructions, but this let us understand what we were doing for the next try.