Purpose:
Isolate cheek cells and see if you have the Alu gene on our number 16 chromosomes.
Materials:
-pipets/pipet tips
-.9% Saline Solution
-waste container
-micro centrifuge
-micro centrifuge tubes
-PCR tubes
-agarose
-1X TAE
-gel chambers + molds
-load dye
-tube racks
-primer mix
-master mix
-water
-positive control DNA
-.9% Saline Solution
-waste container
-micro centrifuge
-micro centrifuge tubes
-PCR tubes
-agarose
-1X TAE
-gel chambers + molds
-load dye
-tube racks
-primer mix
-master mix
-water
-positive control DNA
Procedure:
DNA Preparation
1. Rinse your mouth with salt water to isolate cheek cells in your mouth and then spit it into a cup.
2. Label a 1.5 mL microfuge tube
3. Transfer 1.0mL-1.5 mL of your spit into this tube and centrifuge it; to gather the cells at the bottom of the tube
4. Pour most of the liquid out that is on top of the cells in the tube, leaving only 100 uL on top of the cells
5. Rack the tube to re-suspend the cells
6. Obtain a seperate tube with 5% Chelex
7. Pipet 50 uL of your solution into the Chelex tube
8. Put this Chelex + solution(DNA) tube into a heat block for 10 minutes
9. Take the Chelex + solution(DNA) tube out of the heat block and release the pressure by opening the top
10. Centrifuge for 1 minute
11. Obtain a brand new tube and label DNA with your initials as well
12. Holding your Chelex + solution(DNA) tube at eye level and using a p-200 pipet withdraw 50 uL and put it in the brand new DNA tube
13. Make sure no Chelex beads were transferred
14. Place you tube into a class rack and your teacher will refrigerate it until the PCR portion of the lab
Making Gel
1. 50 mL 1x TAE + 1g agarose and put in microwave till mixed
2. Put into gel tray and let become gel
PCR
1. Obtain a new, tiny PCR tube. Attempt to keep cold for next 3 steps.
2. Pipet 20 uL of Master Mix into it
3. Pipet 20 uL of Primer Mix into it
4. Pipet 10 uL of your from you DNA tube
5. Quick centrifuge
(6.) Controls:
a: 2 students will set up the positive control reactions (+C) for the whole class. There should be + control DNA in the kit. There should be enough for a lane in each gel
b: 2 students will set up negative controls using sterile water. There should be enough for a lane in each gel.
7. Compare your PCR tube to the one the teacher has done, they should have similar amounts of volume in them (50uL)
8. Place this into the thermal cycler and record where you put it
9. The cycling protocol is:
1) 95 C for 2 min
2) 30 cycles of:
-94 C for 30 seconds
-60 C for 30 seconds
-72 C for 2 min
3) 72 C hold for 10 min
4) 4 C hold, infinity
1. Rinse your mouth with salt water to isolate cheek cells in your mouth and then spit it into a cup.
2. Label a 1.5 mL microfuge tube
3. Transfer 1.0mL-1.5 mL of your spit into this tube and centrifuge it; to gather the cells at the bottom of the tube
4. Pour most of the liquid out that is on top of the cells in the tube, leaving only 100 uL on top of the cells
5. Rack the tube to re-suspend the cells
6. Obtain a seperate tube with 5% Chelex
7. Pipet 50 uL of your solution into the Chelex tube
8. Put this Chelex + solution(DNA) tube into a heat block for 10 minutes
9. Take the Chelex + solution(DNA) tube out of the heat block and release the pressure by opening the top
10. Centrifuge for 1 minute
11. Obtain a brand new tube and label DNA with your initials as well
12. Holding your Chelex + solution(DNA) tube at eye level and using a p-200 pipet withdraw 50 uL and put it in the brand new DNA tube
13. Make sure no Chelex beads were transferred
14. Place you tube into a class rack and your teacher will refrigerate it until the PCR portion of the lab
Making Gel
1. 50 mL 1x TAE + 1g agarose and put in microwave till mixed
2. Put into gel tray and let become gel
PCR
1. Obtain a new, tiny PCR tube. Attempt to keep cold for next 3 steps.
2. Pipet 20 uL of Master Mix into it
3. Pipet 20 uL of Primer Mix into it
4. Pipet 10 uL of your from you DNA tube
5. Quick centrifuge
(6.) Controls:
a: 2 students will set up the positive control reactions (+C) for the whole class. There should be + control DNA in the kit. There should be enough for a lane in each gel
b: 2 students will set up negative controls using sterile water. There should be enough for a lane in each gel.
7. Compare your PCR tube to the one the teacher has done, they should have similar amounts of volume in them (50uL)
8. Place this into the thermal cycler and record where you put it
9. The cycling protocol is:
1) 95 C for 2 min
2) 30 cycles of:
-94 C for 30 seconds
-60 C for 30 seconds
-72 C for 2 min
3) 72 C hold for 10 min
4) 4 C hold, infinity