Alu PCR Lab
Purpose:Successfully isolate DNA from cheek cells. Also prepare a PCR reaction for amplification of an Alu insert.
Materials: 9% saline solution, micropipettes, tips wast container, microcentrifuge, microcentrifuge tubes, pcr tubes, agarose, 1xTAE, gel chambers+molds, load dye, chelex, racks, primer mix, master mix, h2o, + control DNA
Procedure:
1.Vigorously swirl 10 mL of saline solution in your mouth for 30 seconds.
2.Expel saline into a cup and swirl to mix the cells.
3.Label a 1.5 mL microfuge tube with you PIN.
4.Transfer 1000 µL to 1500 µL (1 mL to 1.5 mL) of the saline/cell suspension into the labeled microfuge tube.
5. In a microcentrifuge, 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 start over with another 1–1.5 mL 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 100 µL of saline covering it. You may need to add more saline to get up to about 100 µL. 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 50 µL 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 a normal 1.5 mL 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.
11.After heating, gently remove the cap lock and open the tube to release the pressure. Caution: the tube will be hot! Close and then rack or shake the tube well and place it in a centrifuge to spin for 1 minute.
12.Obtain another clean microfuge tube and label it with your PIN. Also write “DNA” on this tube.
13. Holding your tube at eye level, use a P-200 to withdraw 50 µL of supernatant from the Chelex/DNA tube to the new, labeled tube. Be sure NOT to transfer any Chelex beads.
14. Have someone check the “DNA” tube to be sure that no Chelex beads were transferred into it. There should be NO Chelex beads present, as they will interfere with the PCR.
15.Place your DNA tube in the class rack. Your teacher will refrigerate your isolated DNA until you are ready to prepare your PCR amplification.
16.Obtain a tiny PCR tube. Label it with your PIN number, just under the lip.
17.Pipet 20 µL of Master Mix into your PCR tube.
18.Change your pipet tip and add 20 µL of Primer Mix into your PCR tube.
19.With a new pipet tip, add 10 µL of your extracted DNA into your PCR tube.
20.Setting up the controls: a. Two students will be asked to set up the positive control reactions (+C) for the class. They will use the positive control DNA provided in the kit. There should be enough +C PCR sample for one lane on each gel. b. Another two students will set up negative control reactions for the whole class (–C). They will use sterile water. There should be enough –C PCR sample for one lane on each gel.
21.Check the volume of your PCR tube by comparing it to a reference PCR tube with 50 µL in it. It should be near the thermal cycler, set by your teacher.
22.Place your reaction into the thermal cycler and record the location of your tube on the grid provided by your teacher.
23.The cycling protocol for amplification of Alu PV92: 1) 95°C hold for 2 minutes 2) 30 cycles of: 94°C for 30 seconds 60°C for 30 seconds 72°C for 2 minutes 3) 72°C hold for 10 minutes 4) 4°C hold, ∞ infinity
24.Retrieve your PCR tube and place it in a balanced configuration in a microcentrifuge. Spin it briefly (10 seconds) to bring the liquid to the bottom of the reaction tube.
25.Add 5 µL of loading dye to your PCR tube.
26.Carefully load 15 to 20 µL of the DNA/loading dye mixture into a well in your gel. Make sure you keep track of what sample is being loaded into each well.
27.One student (or the instructor) should load 5-10 µL of 100 bp ladder (molecular weight marker) into one of the wells of each gel.
28.When all samples are loaded, attach the electrodes from the gel box to the power supply. Have your teacher check your connections and then electrophorese your samples at 150 Volts for 25–40 minutes.
29.After electrophoresis, the gels will be ready to stain and photograph.
1.Vigorously swirl 10 mL of saline solution in your mouth for 30 seconds.
2.Expel saline into a cup and swirl to mix the cells.
3.Label a 1.5 mL microfuge tube with you PIN.
4.Transfer 1000 µL to 1500 µL (1 mL to 1.5 mL) of the saline/cell suspension into the labeled microfuge tube.
5. In a microcentrifuge, 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 start over with another 1–1.5 mL 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 100 µL of saline covering it. You may need to add more saline to get up to about 100 µL. 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 50 µL 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 a normal 1.5 mL 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.
11.After heating, gently remove the cap lock and open the tube to release the pressure. Caution: the tube will be hot! Close and then rack or shake the tube well and place it in a centrifuge to spin for 1 minute.
12.Obtain another clean microfuge tube and label it with your PIN. Also write “DNA” on this tube.
13. Holding your tube at eye level, use a P-200 to withdraw 50 µL of supernatant from the Chelex/DNA tube to the new, labeled tube. Be sure NOT to transfer any Chelex beads.
14. Have someone check the “DNA” tube to be sure that no Chelex beads were transferred into it. There should be NO Chelex beads present, as they will interfere with the PCR.
15.Place your DNA tube in the class rack. Your teacher will refrigerate your isolated DNA until you are ready to prepare your PCR amplification.
16.Obtain a tiny PCR tube. Label it with your PIN number, just under the lip.
17.Pipet 20 µL of Master Mix into your PCR tube.
18.Change your pipet tip and add 20 µL of Primer Mix into your PCR tube.
19.With a new pipet tip, add 10 µL of your extracted DNA into your PCR tube.
20.Setting up the controls: a. Two students will be asked to set up the positive control reactions (+C) for the class. They will use the positive control DNA provided in the kit. There should be enough +C PCR sample for one lane on each gel. b. Another two students will set up negative control reactions for the whole class (–C). They will use sterile water. There should be enough –C PCR sample for one lane on each gel.
21.Check the volume of your PCR tube by comparing it to a reference PCR tube with 50 µL in it. It should be near the thermal cycler, set by your teacher.
22.Place your reaction into the thermal cycler and record the location of your tube on the grid provided by your teacher.
23.The cycling protocol for amplification of Alu PV92: 1) 95°C hold for 2 minutes 2) 30 cycles of: 94°C for 30 seconds 60°C for 30 seconds 72°C for 2 minutes 3) 72°C hold for 10 minutes 4) 4°C hold, ∞ infinity
24.Retrieve your PCR tube and place it in a balanced configuration in a microcentrifuge. Spin it briefly (10 seconds) to bring the liquid to the bottom of the reaction tube.
25.Add 5 µL of loading dye to your PCR tube.
26.Carefully load 15 to 20 µL of the DNA/loading dye mixture into a well in your gel. Make sure you keep track of what sample is being loaded into each well.
27.One student (or the instructor) should load 5-10 µL of 100 bp ladder (molecular weight marker) into one of the wells of each gel.
28.When all samples are loaded, attach the electrodes from the gel box to the power supply. Have your teacher check your connections and then electrophorese your samples at 150 Volts for 25–40 minutes.
29.After electrophoresis, the gels will be ready to stain and photograph.