Alu Polymerase Chain Reaction (PCR) Lab:
Objectives:
1. Successfully isolate DNA from cheek cells. 2. Prepare a PCR reaction for amplification of an Alu insert. |
Materials:
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Procedure:
DNA Preparation Using a Saline Mouthwash
1. Swirl 10 mL of saline solution (salt water) in your mouth for 30 seconds and spit into a cup.
2. Transfer 1 mL of the saline/cheek cell suspension into a labeled 1.5 mL microfuge tube. Suspend in the microcentrifuge for 1 minute.
3. Pour the supernatant out into your cup leaving 0.1 mL covering the cheek cell pellet at the bottom of the microfuge tube.
4. Put 0.05 mL of your cell suspension into a labelled tube of Chelex from your teacher. Place that tube into theheat block for 10 minutes.
5. After 10 minutes, place the tube in a centrifuge for 1 minute.
6. Take out 0.05 mL of the supernatant from the tube with a P-200 pipette and transfer to a new microfuge tube labelled ¨DNA¨. Ensure there are NO Chelex beads. Place the DNA tube on the class rack to be refrigerated until PCR amplification.
Polymerase Chain Reaction (PCR)
1. Pipette 0.02 mL of Master Mix into your labelled PCR tube.
2. Change your pipette tip and add 0.02 mL of Primer Mix into your PCR tube.
3. With a new pipette tip, add 0.01 mL of your isolated DNA into your PCR tube.
4. Create a positive control with the provided positive DNA sample, and create a negative control with sterile water.
5. Place the PCR tube in the thermal cycler where it will undergo denaturation, annealing, and extension.
Agarose Gel Electrophoresis of Amplified DNA
1. Calculate the mass of agarose needed for 50 mL total volume of agarose solution.
C1V1=C2V2
2. Mix 50 mL of 1X TAE and 1 g agarose together in a beaker. Heat until dissolved.
3. Pour in mold when clear.
4. Spin your PCR tube in a microcentrifuge for 10 seconds.
5. Add 0.004 mL of loading dye to your PCR tube.
6. Load 0.02 mL of DNA/loading dye mixture into a well of your agarose gel.
7. Load one of the wells with the 100 bp ladder into one of the wells of each gel.
8. Once all samples are loaded, attach electrodes from the gel box to the power supply. Make sure the DNA is going negative to positive since it has a negative charge. Electrophorese at 150 Volts for 40 minutes.
9. After electrophoresis, place the agarose gel in a staining tray.
10. Pour enough ethidium bromide to cover the gel and wait for 20 minutes.
11. Pour the ethidium bromide back into its storage bottle. Then pour enough water in the staining tray to cover the gel and wait for 5 minutes.
12. Pour the water out of the staining tray into a hazardous waste container and place the stained gel on a UV light box. Dispose hazardous material properly.
13. Place the camera over the gel and take a photograph.
DNA Preparation Using a Saline Mouthwash
1. Swirl 10 mL of saline solution (salt water) in your mouth for 30 seconds and spit into a cup.
2. Transfer 1 mL of the saline/cheek cell suspension into a labeled 1.5 mL microfuge tube. Suspend in the microcentrifuge for 1 minute.
3. Pour the supernatant out into your cup leaving 0.1 mL covering the cheek cell pellet at the bottom of the microfuge tube.
4. Put 0.05 mL of your cell suspension into a labelled tube of Chelex from your teacher. Place that tube into theheat block for 10 minutes.
5. After 10 minutes, place the tube in a centrifuge for 1 minute.
6. Take out 0.05 mL of the supernatant from the tube with a P-200 pipette and transfer to a new microfuge tube labelled ¨DNA¨. Ensure there are NO Chelex beads. Place the DNA tube on the class rack to be refrigerated until PCR amplification.
Polymerase Chain Reaction (PCR)
1. Pipette 0.02 mL of Master Mix into your labelled PCR tube.
2. Change your pipette tip and add 0.02 mL of Primer Mix into your PCR tube.
3. With a new pipette tip, add 0.01 mL of your isolated DNA into your PCR tube.
4. Create a positive control with the provided positive DNA sample, and create a negative control with sterile water.
5. Place the PCR tube in the thermal cycler where it will undergo denaturation, annealing, and extension.
Agarose Gel Electrophoresis of Amplified DNA
1. Calculate the mass of agarose needed for 50 mL total volume of agarose solution.
C1V1=C2V2
2. Mix 50 mL of 1X TAE and 1 g agarose together in a beaker. Heat until dissolved.
3. Pour in mold when clear.
4. Spin your PCR tube in a microcentrifuge for 10 seconds.
5. Add 0.004 mL of loading dye to your PCR tube.
6. Load 0.02 mL of DNA/loading dye mixture into a well of your agarose gel.
7. Load one of the wells with the 100 bp ladder into one of the wells of each gel.
8. Once all samples are loaded, attach electrodes from the gel box to the power supply. Make sure the DNA is going negative to positive since it has a negative charge. Electrophorese at 150 Volts for 40 minutes.
9. After electrophoresis, place the agarose gel in a staining tray.
10. Pour enough ethidium bromide to cover the gel and wait for 20 minutes.
11. Pour the ethidium bromide back into its storage bottle. Then pour enough water in the staining tray to cover the gel and wait for 5 minutes.
12. Pour the water out of the staining tray into a hazardous waste container and place the stained gel on a UV light box. Dispose hazardous material properly.
13. Place the camera over the gel and take a photograph.