Purpose:
To use our own DNA to see which alu alleles we have. Successfully isolate DNA from cheek cells. Prepare a PCR reaction for amplification of an Alu insert.
To use our own DNA to see which alu alleles we have. Successfully isolate DNA from cheek cells. Prepare a PCR reaction for amplification of an Alu insert.
Materials:
0.9% saline solution micro-pipettes, tips waste container micro-centrifuge, tubes PCR tubes agarose water |
1x TAE gel chambers and molds load dye chelex racks primer mix master mix positive 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 microcentrifuge 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 1x TAE 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.
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 microcentrifuge 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 1x TAE 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.