Hypothesis: I have a feeling that this lab is not going to get accurate results because it seems very unorganized and there are too many steps for us to not mess up. Purpose: To determine the frequency of the Alu insert in the Stem Bio class using PCR Materials:
Cheek cells
Chelex
Master mix
Bleach
Primer mix
TBE buffer
Agrose gel
DNA stain solution.
In this lab we looked at our DNA, we messed it up once so we had to do it again, and im a homozygos. Procedure:
Swirl 10 mL of 0.9% NaCl solution in your mouth for 30 seconds.
Expel saline into cup and swirl the mix
Label a 1.5 mL microfuge tube with your initials
Transfer 1 mL of the saline solution containing your cells into the microfuge tube
In a Microcentrifuge, spin your saline cell suspention for 1 minute at 10,000 RPM to pellet the cells.
Observe the cell pellet at the bottom of the tube. If you do not have one repeat the steps above till you do.
Add or subtract Saline solution to get the covering to about 100 µL
Rack the tube
Obtain a tube of 5% Chelex; Label it.
Withdraw 50 µL from the saline cell/solution. Add it to the Chelex.
Slide a cap lock on
Place it in a heat block fir 10 Min.
Remove the cap lock.
Rack the tube.
Centrifuge for 1 min.
Obtain a clean tube and label it with your initials and "DNA".
Withdraw 50 µL of the superintendent in the Chelex tube, and place in in the DNA tube.
Refrigerate until next step
Obtain a tiny PCR tube. Label it with your initials.
Add 20 µL of Master Mix into the PCR tube.
Add 20 µL of the primer mix into the PCR tube.
Add 10 µL of your extracted DNA into the tube.
Place in the thermal cycler.
Results: It turned out that I am homozygous. This means that I have two of the same alleles rather than two different ones.
Conclusion: We messed up the lab pretty bad and had to restart the lab, but I still enjoyed the process and I think I learned a lot about using pipettes. I did not particularly enjoy putting salt water in my mouth, but the rest of the lab was fun.