Alu PCR Lab
Purpose: To isolate DNA from our cheek cells and create a PCR reaction to see if we are homozygous or heterozygous.
Materials:
- 0.9% saline solution
- Micropipettes, tips
- waste container
- microcentrifuge
- microcentrifuge tubes
- PCR tubes
- agarose
- 1XTAE
- loading dye
- gel chambers and molds
- chelex
- racks
- primer mix
- master mix
- water and control DNA
Procedure:
1. Swirl 10 ml of saline solution in your mouth for 30 seconds. Expel saline into a cup and swirl to mix cells.
2. Transfer 1000 microliters of the saline/cell suspension into your labeled microfuge tube. Spin in a microcentrifuge to pellet the cells. Pour off the supernatent, allowing 100 microliters to cover the cell pallet. Rack the sample.
3. Withdraw 50 microliters of your cell suspension and add it to a tube containing Chelex.
4. Apply Chelex tube to a heat block for 10 minutes.
5. Remove Chelex tube from heat block. Use a P-200 to withdraw 50 microliters of supernatent from the Chelex tube and transfer to a fresh tube.
6. Obtain a tiny PCR tube and keep on ice.
7. Pipette 20 microliters of Master Mix into the PCR tube. Then add 20 microliters of Primer Mix.
8. Add 10 microliters of your extracted DNa into the PCR tube.
9. Place reaction into a thermal cycler.
10. Retrieve PCR tube and spin in a microcentrifuge. Then, add 5 microliters of loading dye.
11. Create and pour gels. Add 1XTAE solution.
12. Load 15 to 20 microliters of the DNA/loading dye mixture into a well in your gel.
13. Load 5 to 10 microliters of a 100 base-pair ladder (molecular weight marker) into the one well in each gel for later comparison.
14. Run gels.
Results: My results are in a lane 7
Materials:
- 0.9% saline solution
- Micropipettes, tips
- waste container
- microcentrifuge
- microcentrifuge tubes
- PCR tubes
- agarose
- 1XTAE
- loading dye
- gel chambers and molds
- chelex
- racks
- primer mix
- master mix
- water and control DNA
Procedure:
1. Swirl 10 ml of saline solution in your mouth for 30 seconds. Expel saline into a cup and swirl to mix cells.
2. Transfer 1000 microliters of the saline/cell suspension into your labeled microfuge tube. Spin in a microcentrifuge to pellet the cells. Pour off the supernatent, allowing 100 microliters to cover the cell pallet. Rack the sample.
3. Withdraw 50 microliters of your cell suspension and add it to a tube containing Chelex.
4. Apply Chelex tube to a heat block for 10 minutes.
5. Remove Chelex tube from heat block. Use a P-200 to withdraw 50 microliters of supernatent from the Chelex tube and transfer to a fresh tube.
6. Obtain a tiny PCR tube and keep on ice.
7. Pipette 20 microliters of Master Mix into the PCR tube. Then add 20 microliters of Primer Mix.
8. Add 10 microliters of your extracted DNa into the PCR tube.
9. Place reaction into a thermal cycler.
10. Retrieve PCR tube and spin in a microcentrifuge. Then, add 5 microliters of loading dye.
11. Create and pour gels. Add 1XTAE solution.
12. Load 15 to 20 microliters of the DNA/loading dye mixture into a well in your gel.
13. Load 5 to 10 microliters of a 100 base-pair ladder (molecular weight marker) into the one well in each gel for later comparison.
14. Run gels.
Results: My results are in a lane 7
Analysis: I found out I am homozygous negative because there was only one bar and it was at the lower point of the sample.
Conclusion: We were able to fulfill our purpose in this project. We completed every step and were able to isolate our DNA and copy the Alu gene. From this we were able to find out if we were heterozygous or homozygous.
In our process we could have made errors pipetting our DNA into the gel and extracting the DNA from our cheeks. Our group did a good job staying on task and getting through the project quickly and successfully.
Conclusion: We were able to fulfill our purpose in this project. We completed every step and were able to isolate our DNA and copy the Alu gene. From this we were able to find out if we were heterozygous or homozygous.
In our process we could have made errors pipetting our DNA into the gel and extracting the DNA from our cheeks. Our group did a good job staying on task and getting through the project quickly and successfully.