Alu PCR Lab
Purpose:
Determine the genome type for the alu insert. Determine the frequency of the Alu.
Determine the genome type for the alu insert. Determine the frequency of the Alu.
Materials:
-Ependorfs
-Pipets (P-20 and P-200)
-Agarose gel
- 0.97 Saline solution
-Pipet tips
-Microwave
- Microcenterfuge
-Heat block
-Ice
-Primer Mix
-Master Mix (nuclotides, DNA polymerase, buffer)
- 5.7 Chelex
-Tracking dye
-DNA stain "gel red"
-Positive Control DNA
- TBE Buffer
-Cheek cells
-Base pair ladder
-Ependorfs
-Pipets (P-20 and P-200)
-Agarose gel
- 0.97 Saline solution
-Pipet tips
-Microwave
- Microcenterfuge
-Heat block
-Ice
-Primer Mix
-Master Mix (nuclotides, DNA polymerase, buffer)
- 5.7 Chelex
-Tracking dye
-DNA stain "gel red"
-Positive Control DNA
- TBE Buffer
-Cheek cells
-Base pair ladder
Procedure:
1) Swirl 10mL of saline solution in your mouth for 30 seconds.
2) Spit saline into the cup and swirl it to mix the cells.
3) Label a 1.5 mL mircrofuge with your initials
4) Transfer 1mL to 1.5 mL of the saline/cell suspension into the mircrofuge.
5) Spin your saline cell suspension in a mircocenterfuge for one minute to pellet the cells. Use another sample as a balance
6) Pour off the supernatant into your cup. Make sure not to lose the cell pellet.
7) Make sure there is about 100 uL of saline covering it. Rack the tube to resuspend the cell.
8) Obtain a tube of Chelex and label it with your initials
9) Withdraw 50 uL of cell suspension from step 7 and add it to the tube of Chelex
10) Slide the cap lock onto the tube lid and place it on the heat block for 10 minutes.
11) Remove the cap lock and open the tube to release the pressure. Then, close it and rack the tube. Put it in the centrifuge to spin for one minute.
12) Obtain another microfuge and label it with your intials
13) Use a p-200 to transfer 50 uL of supernatant from the Chelex tube into the new, labeled tube, making sure not to get the Chelex beads.
14) Obtain a tiny PCR tube and label it with your intials.
15) Pipet 20uL of Master Mix into the PCR tube.
16) Change the pipet tip and add 20 uL of Primer Mix into the PCR tube.
17) Change pipet tip and add 20 uL of extracted DNA into your PCR tube.
Results:
My result was two bands. I had a heterozygous +/-
There were bands at 715 and 415.
My result was two bands. I had a heterozygous +/-
There were bands at 715 and 415.
Conclusion:
There was one thing I wish I had done differently. The bands on the gel were not very clear. Next time, I will place the liquid in the gel more carefully, to get more accurate and clear results. I learned how to use pipets and gained knowledge on the Alu gene.
There was one thing I wish I had done differently. The bands on the gel were not very clear. Next time, I will place the liquid in the gel more carefully, to get more accurate and clear results. I learned how to use pipets and gained knowledge on the Alu gene.