Alu PCR Lab
Purpose of the Lab:
- List and explain the importance of each component of PCR
- Compare PCR to cellular DNA replication
- Associate the temperature changes with the cycling steps of PCR
- Succesfully isolate DNA from cheek cells
- Prepare a PCR reaction for amplication of an Alu insert
Materials:
.9% saline solution
Micropipettes, tips
Waste Container
Microcentrifuge
Microcentifuge tubes
PCR tubes
Agarose
1XTAE
Gel chambers and molds
Load dye
Chelex
Racks
Primer Mix
Master Mix
Water
Positive control DNA
Procedure:
1. Swirl 10mL of saline solution in your mouth for 30 seconds.
2. Expel saline into a cup and swirl to mix the cells.
3. Label a 1.5mL microfuge tube with your PIN.
4. Transfer 1000uL(1mL to 1.5mL) of the saline/cell suspension into the labeled microfuge tubes.)
5. In a microcentifuge, spin your saline cell suspension for 1 minute to pellet the cells. Be sure to use another student's sample as a balance.
6. Observe our cell pellet at the bottom of the tube. If you do not have one, you may need to startr over with another 1-1.5mL of saline rinse. Pour off the supernatant into your cup, being careful NOT to lose your cell pellet.
7. Check to make sure you can see your cell pellet and that there is about 100uL of saline covering it. You may need to add more saline to get up to about 100uL.
Rack or flick tube to mix, which will "resuspend" the cell and make an evenly mixed solution.
8. Obtain a tube of Chelex from your instructor. Label with your PIN.
9. Withdraw 50uL of your cell suspension from step 7 and add it to the tube containing Chelex.
10. Heat Block Version: If your Chelex(with the cell suspension) is in normal 1.5mL microfuge tube, take your tube to a heat block station. Slide a cap lock onto the tube lid and place it in the heat block for 10 minutes. Keep track of your tube in the heat block.
PCR tube version:
- List and explain the importance of each component of PCR
- Compare PCR to cellular DNA replication
- Associate the temperature changes with the cycling steps of PCR
- Succesfully isolate DNA from cheek cells
- Prepare a PCR reaction for amplication of an Alu insert
Materials:
.9% saline solution
Micropipettes, tips
Waste Container
Microcentrifuge
Microcentifuge tubes
PCR tubes
Agarose
1XTAE
Gel chambers and molds
Load dye
Chelex
Racks
Primer Mix
Master Mix
Water
Positive control DNA
Procedure:
1. Swirl 10mL of saline solution in your mouth for 30 seconds.
2. Expel saline into a cup and swirl to mix the cells.
3. Label a 1.5mL microfuge tube with your PIN.
4. Transfer 1000uL(1mL to 1.5mL) of the saline/cell suspension into the labeled microfuge tubes.)
5. In a microcentifuge, spin your saline cell suspension for 1 minute to pellet the cells. Be sure to use another student's sample as a balance.
6. Observe our cell pellet at the bottom of the tube. If you do not have one, you may need to startr over with another 1-1.5mL of saline rinse. Pour off the supernatant into your cup, being careful NOT to lose your cell pellet.
7. Check to make sure you can see your cell pellet and that there is about 100uL of saline covering it. You may need to add more saline to get up to about 100uL.
Rack or flick tube to mix, which will "resuspend" the cell and make an evenly mixed solution.
8. Obtain a tube of Chelex from your instructor. Label with your PIN.
9. Withdraw 50uL of your cell suspension from step 7 and add it to the tube containing Chelex.
10. Heat Block Version: If your Chelex(with the cell suspension) is in normal 1.5mL microfuge tube, take your tube to a heat block station. Slide a cap lock onto the tube lid and place it in the heat block for 10 minutes. Keep track of your tube in the heat block.
PCR tube version: