Alu pcr lab
Purpose: To determine the frequency of the Alu insert in the STEM Bio class using PCR.
Hypothesis: I think that I am a heterozygeous.
Materials:
Cheek Cell
Chelex
Master Mix: nucleotides, DNA polymerase
Prime Mix
TBE: Buffer, Agorose Gel
DNA Stain Solution
Centrifuge
Tube rack
Reaction Tubes
Permanent Markers
Pack of 4 Colors
Micropipets
Micropipet Tips
Sterile Tubes
Plastic Beaker
Gel Box
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 /cell suspension 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 50 ul 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 tiny 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. Retrieve 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, the gels will be ready to stain and photograph.
Results-When we did it for the third time, i finally got a result. It turns out that I'm (-,+). My result is in lane 4, (the fourth one from the left hand side.)
Analysis- The mistake we made the first day, which then forced us to restart the experiment the second day, was honestly just something that we have to learn from. As a class we learned the importance of reading directions closely and not going too quickly. By doing the lab twice, we had a bettering understanding of the material. Ms. Flasher took a picture of the DNA gels over the weekend, so we tried our best to analyze the picture.
Hypothesis: I think that I am a heterozygeous.
Materials:
Cheek Cell
Chelex
Master Mix: nucleotides, DNA polymerase
Prime Mix
TBE: Buffer, Agorose Gel
DNA Stain Solution
Centrifuge
Tube rack
Reaction Tubes
Permanent Markers
Pack of 4 Colors
Micropipets
Micropipet Tips
Sterile Tubes
Plastic Beaker
Gel Box
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 /cell suspension 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 50 ul 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 tiny 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. Retrieve 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, the gels will be ready to stain and photograph.
Results-When we did it for the third time, i finally got a result. It turns out that I'm (-,+). My result is in lane 4, (the fourth one from the left hand side.)
Analysis- The mistake we made the first day, which then forced us to restart the experiment the second day, was honestly just something that we have to learn from. As a class we learned the importance of reading directions closely and not going too quickly. By doing the lab twice, we had a bettering understanding of the material. Ms. Flasher took a picture of the DNA gels over the weekend, so we tried our best to analyze the picture.