pcr lab procedure:
To successfully isolate DNA from our cheek cells and to prepare a PCR reaction for the amplification of an ALU insert.
PCR LAB Materials:
- .9% Saline Solution
- Dixie Cups
- Micro pipettes
- Micro pipette tips
- waste container
- Microcentrifuge
- Microcentrifuge tubes
- PCR tubes
- Agarose
- 1 x TAE
- gel chambers & molds
- load dye
- chelex
- racks
- primer mix
- master mix
- Water
- + control DNA
- Dixie Cups
- Micro pipettes
- Micro pipette tips
- waste container
- Microcentrifuge
- Microcentrifuge tubes
- PCR tubes
- Agarose
- 1 x TAE
- gel chambers & molds
- load dye
- chelex
- racks
- primer mix
- master mix
- Water
- + control DNA
pca lab procedure:
1. Swish 10 mL. (milliliters) of saline solution in your mouth to extract cheeks cells.
2. Spit solution in dixie cup. Swirl cup to mix the cells
3. Label a 1.5 mL. microfuge tube with a customized PIN
4. Transfer 1000 uL. (microliters) of saline/cell suspension into labeled microfuge tube.
5. Spin suspension, in microfuge, for 1 minute to pellet cells at bottom. Empty tube of saline, leaving approximately 100 ul. saline on top
6. Add 50 ul. cell suspension to microfuge tube of chelex beads to remove waste.
7. Heat chelex for 10 minutes at 99 C.
8. Release pressure from tube then rack well and centrifuge for 1 minute.
9. Obtain 2nd clean microfuge tube and label with same PIN and DNA.
10. Withdraw 50 uL. of supernatant from chelex/DNA tube to new tube.
11. Obtain small PCR tube. Label with PIN.
12. Pipet 20 uL. of Master Mix into PCR tube. With new tip, add 20 uL. Primer Mix to same tube.
13. Using new tip, add 10 uL. of previously extracted DNA to PCR tube.
14. Thermal Cycle for specified time.
15. Prepare electrophoresis gel.
16. Add 1 g, agarose powder to 50 mL. 1 x TAE
17. Heat and stir alternately to dissolve agarose.
18. Pour dissolved mixture into mold and let cool until solid.
19. Remove PCR tubes fro thermal cycler and spin in microcentrifuge for 10 seconds.
20. Add 5 uL. load dye to PCR tube. Centrifuge for 10 seconds.
21. CAREFULLY load 20 uL. DNA/load dye mixture into well of gel.
2. Spit solution in dixie cup. Swirl cup to mix the cells
3. Label a 1.5 mL. microfuge tube with a customized PIN
4. Transfer 1000 uL. (microliters) of saline/cell suspension into labeled microfuge tube.
5. Spin suspension, in microfuge, for 1 minute to pellet cells at bottom. Empty tube of saline, leaving approximately 100 ul. saline on top
6. Add 50 ul. cell suspension to microfuge tube of chelex beads to remove waste.
7. Heat chelex for 10 minutes at 99 C.
8. Release pressure from tube then rack well and centrifuge for 1 minute.
9. Obtain 2nd clean microfuge tube and label with same PIN and DNA.
10. Withdraw 50 uL. of supernatant from chelex/DNA tube to new tube.
11. Obtain small PCR tube. Label with PIN.
12. Pipet 20 uL. of Master Mix into PCR tube. With new tip, add 20 uL. Primer Mix to same tube.
13. Using new tip, add 10 uL. of previously extracted DNA to PCR tube.
14. Thermal Cycle for specified time.
15. Prepare electrophoresis gel.
16. Add 1 g, agarose powder to 50 mL. 1 x TAE
17. Heat and stir alternately to dissolve agarose.
18. Pour dissolved mixture into mold and let cool until solid.
19. Remove PCR tubes fro thermal cycler and spin in microcentrifuge for 10 seconds.
20. Add 5 uL. load dye to PCR tube. Centrifuge for 10 seconds.
21. CAREFULLY load 20 uL. DNA/load dye mixture into well of gel.