Purpose
Student should be able to:
1. List and explain the importance of each component of PCR.
2. Compare PCR to cellular DNA replication.
3. Associate the temperature changes with the cycling steps of PCR.
4. Successfully isolate DNA from cheek cells
5. Prepare a PCR reaction for amplification of an Alu insert.
1. List and explain the importance of each component of PCR.
2. Compare PCR to cellular DNA replication.
3. Associate the temperature changes with the cycling steps of PCR.
4. Successfully isolate DNA from cheek cells
5. Prepare a PCR reaction for amplification of an Alu insert.
Materials
.9% saline solution
micropipet tips
waste container
microcentrifuge
microcentrifuge tubes
PCR tubes
agarose
IXTAE
gel chambers + molds
load dye
chelex
racks
primer mix
master mix
water
control DNA
micropipet tips
waste container
microcentrifuge
microcentrifuge tubes
PCR tubes
agarose
IXTAE
gel chambers + molds
load dye
chelex
racks
primer mix
master mix
water
control DNA
procedure
DNA Preparation
1. swirl 10 mL of saline
2. spit saline into cup and swirl to mix cells
3. label 1.5 mL microcentrifuge with pin
4. transfer 1000 mL of saline solution into microcentrifuge tube
5. spin tube to form pellet of cells
6. pour supernatant out
7. leave 100 mL of saline covering cells
8. obtain and label a tube of chelex
9. Transfer 50 mL of solution into chelex tube
10. move solution to heat block and heat for 10 mins
11.release pressure from tube
12. Label a new microcentrifuge tube "DNA"
13. withdraw 50 mL of supernatant from chelex/DNA tube
14. make sure no chelex beads were transferred
15. refrigerate DNA tube until next day
Polymerase Chain Reaction
1. label PCR tube with pin number
2. pipet 20 mL of master mix into PCR tube
3. add 20 mL of primer mix into PCR tube
4. add 10 mL of DNA into PCR tube
5.
1. swirl 10 mL of saline
2. spit saline into cup and swirl to mix cells
3. label 1.5 mL microcentrifuge with pin
4. transfer 1000 mL of saline solution into microcentrifuge tube
5. spin tube to form pellet of cells
6. pour supernatant out
7. leave 100 mL of saline covering cells
8. obtain and label a tube of chelex
9. Transfer 50 mL of solution into chelex tube
10. move solution to heat block and heat for 10 mins
11.release pressure from tube
12. Label a new microcentrifuge tube "DNA"
13. withdraw 50 mL of supernatant from chelex/DNA tube
14. make sure no chelex beads were transferred
15. refrigerate DNA tube until next day
Polymerase Chain Reaction
1. label PCR tube with pin number
2. pipet 20 mL of master mix into PCR tube
3. add 20 mL of primer mix into PCR tube
4. add 10 mL of DNA into PCR tube
5.