Purpose, Materials, and Procedure
Purpose:
The purpose of this lab is to observe our individual genotype using strands of DNA isolated from our cheek cells. We should also demonstrate the ability to successfully isolate DNA from cheek cells and prepare a PCR reaction for the amplification of an alu insert.
Materials:
0.9% Saline Solution
Micropipets and tips
Waste container
Microcentrifuge and microcentrifuge tubes
PCR tubes
Agarose
1XTAE
Loading dye
Gel chambers and molds
Chelex
Racks
Primer and master mixes
Water and control DNA
Thermal cycler
Procedure:
1. Robustly swish 10 mL of the saline solution in your mouth for 30 seconds. Expel into a cup and swirl to mix the cells
2. After labeling a 1.5 mL microfuge tube, transfer 1 mL of the saline/cell suspension into the tube
3. Spin the saline cell suspension in a microcentrifuge for one minute and observe the cell pellet
4. Transfer the cell pellet into a new tube with 100 uL of saline
5. After receiving a tube of chelex, transfer 50 uL of the cell suspension from the original tube into the chelex tube
6. Close the tube and place it in a heat block for ten minutes
7. Remove the tube and open it to release the pressure that had built up
8. Obtain another microfuge tube and transfer 50 uL of the chelex/DNA to the new tube DO NOT TRANSFER ANY CHELEX BEADS
9. Check to make sure there are no chelex beads and place it in the class rack
10. Refrigerate until prepared for PCR amplification
11. Obtain a tiny PCR tube
12. Pipet 20 uL of the master mix in the PCR tube
13. Using a new tip, pipet 20 uL of the the primer mix into the PCR tube
14. Add 10 uL of your extracted DNA into the PCR tube
15. Place the reaction into the thermal cycler and record the location of your tube
16. Retrieve the PCR tube and spin it in a microcentrifuge for about ten seconds
17. Add 5 uL of the loading dye to the PCR tubes
18. Carefully load 15-20 uL of the misture into a well in your gel.
19. The instructor will load 5-10 uL of 100 bp ladder into the wells of each gel
20. When all the gels are loaded, attach the electrodes from the gel box to the power supply
21. Electrophorese your samples at 150 Volts for 25-40 minutes
22. Stain and photograph the gels and record the results
The purpose of this lab is to observe our individual genotype using strands of DNA isolated from our cheek cells. We should also demonstrate the ability to successfully isolate DNA from cheek cells and prepare a PCR reaction for the amplification of an alu insert.
Materials:
0.9% Saline Solution
Micropipets and tips
Waste container
Microcentrifuge and microcentrifuge tubes
PCR tubes
Agarose
1XTAE
Loading dye
Gel chambers and molds
Chelex
Racks
Primer and master mixes
Water and control DNA
Thermal cycler
Procedure:
1. Robustly swish 10 mL of the saline solution in your mouth for 30 seconds. Expel into a cup and swirl to mix the cells
2. After labeling a 1.5 mL microfuge tube, transfer 1 mL of the saline/cell suspension into the tube
3. Spin the saline cell suspension in a microcentrifuge for one minute and observe the cell pellet
4. Transfer the cell pellet into a new tube with 100 uL of saline
5. After receiving a tube of chelex, transfer 50 uL of the cell suspension from the original tube into the chelex tube
6. Close the tube and place it in a heat block for ten minutes
7. Remove the tube and open it to release the pressure that had built up
8. Obtain another microfuge tube and transfer 50 uL of the chelex/DNA to the new tube DO NOT TRANSFER ANY CHELEX BEADS
9. Check to make sure there are no chelex beads and place it in the class rack
10. Refrigerate until prepared for PCR amplification
11. Obtain a tiny PCR tube
12. Pipet 20 uL of the master mix in the PCR tube
13. Using a new tip, pipet 20 uL of the the primer mix into the PCR tube
14. Add 10 uL of your extracted DNA into the PCR tube
15. Place the reaction into the thermal cycler and record the location of your tube
16. Retrieve the PCR tube and spin it in a microcentrifuge for about ten seconds
17. Add 5 uL of the loading dye to the PCR tubes
18. Carefully load 15-20 uL of the misture into a well in your gel.
19. The instructor will load 5-10 uL of 100 bp ladder into the wells of each gel
20. When all the gels are loaded, attach the electrodes from the gel box to the power supply
21. Electrophorese your samples at 150 Volts for 25-40 minutes
22. Stain and photograph the gels and record the results