Alu PCR Lab
Purpose
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
Successfully isolate DNA from cheek cells
Prepare a PCR reaction for amplification of an Alu insert
Materials
.9& saline solution
Micro pipettes, tips
Waste container
Microcentifuge
Microcentrifuge tubes
PCR tubes
Agarose
Racks
Primer mix
1X TAE
Gel chambers and molds
Load due
Master mix
Water
Positive control DNA
Chelex
Procedure
1. Violently swish salt water in mouth for 30 seconds
2. Spit out salt water
3. Create a PIN number and label 1.5mL microfuge tube with it
4. Transfer 1mL to 1.5mL to the saline suspension into the labeled PIN microfuge tube
5. Spin tube in microcentrifuge for 1 minute
6. Pour remaining supernatant in sink or cup
7. Rack or scrape tube on the rack to resuspend the cell
8. Get 5% Chelex and label it
9. Take out 50ul of the suspended cells and add it to Chelex tube
10. Put the Chelex solution on a heat block section for 10 minutes and set it to 99 degrees Celsius
11. Take it out of heat block and open the tube to release the pressure inside of it
12. Get another microfuge tube and label it with PIN number and write DNA
13. Use P-200 to take out 50 ul of supernatant from DNA tube to the new tube you just labeled
14. Rack DNA tube
15. Get tiny PCR tube
16. Pipet 20ul of Master Mix in the small tube
17. Also add 20ul of extracted DNA into PCR tube
18. Place small tube in thermal cycler
19. Then make the gel, and you get 50ml of 1X TAE and 1 gram of agarose
20. Heat until agarose dissolved
21. Pour into mold and wait until it is cool
22.After a day get the PCR tube and put it in the centrifuge
23. Add 20ul sample to a fresh 1.5ml tube
24. Add 4ul of load dye
25. Then centrifuge it again
26. Load 20ul in my well
27. Record the gel number and lane
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
Successfully isolate DNA from cheek cells
Prepare a PCR reaction for amplification of an Alu insert
Materials
.9& saline solution
Micro pipettes, tips
Waste container
Microcentifuge
Microcentrifuge tubes
PCR tubes
Agarose
Racks
Primer mix
1X TAE
Gel chambers and molds
Load due
Master mix
Water
Positive control DNA
Chelex
Procedure
1. Violently swish salt water in mouth for 30 seconds
2. Spit out salt water
3. Create a PIN number and label 1.5mL microfuge tube with it
4. Transfer 1mL to 1.5mL to the saline suspension into the labeled PIN microfuge tube
5. Spin tube in microcentrifuge for 1 minute
6. Pour remaining supernatant in sink or cup
7. Rack or scrape tube on the rack to resuspend the cell
8. Get 5% Chelex and label it
9. Take out 50ul of the suspended cells and add it to Chelex tube
10. Put the Chelex solution on a heat block section for 10 minutes and set it to 99 degrees Celsius
11. Take it out of heat block and open the tube to release the pressure inside of it
12. Get another microfuge tube and label it with PIN number and write DNA
13. Use P-200 to take out 50 ul of supernatant from DNA tube to the new tube you just labeled
14. Rack DNA tube
15. Get tiny PCR tube
16. Pipet 20ul of Master Mix in the small tube
17. Also add 20ul of extracted DNA into PCR tube
18. Place small tube in thermal cycler
19. Then make the gel, and you get 50ml of 1X TAE and 1 gram of agarose
20. Heat until agarose dissolved
21. Pour into mold and wait until it is cool
22.After a day get the PCR tube and put it in the centrifuge
23. Add 20ul sample to a fresh 1.5ml tube
24. Add 4ul of load dye
25. Then centrifuge it again
26. Load 20ul in my well
27. Record the gel number and lane