PCR Lab write-up.
This lab was purpose was to figure out the percentage of people in our class that have the Aulle insert in their DNA.
Hypothesis:
I predict that about 40% of the class will have at least one part of the gene mutation Alllue. because it is a mutation, it is likely that there where more people with it because
I predict that about 40% of the class will have at least one part of the gene mutation Alllue. because it is a mutation, it is likely that there where more people with it because
Materials:
- 0.97% saline solution
- My cheek cells
- 5% chelex solution
- Primer mix
- Master mix
- TBE buffer
- Agaros gel
- Tracking Dye
- DNA stain
- Positive control DNA
- Base Pair Ladder
- P-20, P-200, and P-1000 pipit
- Cap Locks
- Ice
- Eppendorf test tubes
- Micro-Centrifuge
- Heat block
- THermal cycler
- Microwave
- Heat gloves
- Graduated cylinder
- Flask
- Gel Block
- UV Light Box
- Electricity
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.
I did not have the Alu segment in my DNA, so I am homozygous. There was an even split between homozygous and heterozygous students.
Analysis:
Neither my mother nor my father passed the Alu repeat in their DNA. If one of them had it, there would have been a 415 bp fragment, and an 715 bp fragment. If both of them had it, there would only be a fragment at 715 bp. In my case, I only had a fragment at 415 bp, so neither of my parents has the Alu repeat.
Conclusion:
The lab was really about learning how to use lab equipment. We got to practice using laboratory materials such as centrifuges, microfuge tubes, and micro pipets. Overall, this was a great way to learn about safe lab practices, proper use of equipment (balancing the centrifuge), and PCR.
Analysis:
Neither my mother nor my father passed the Alu repeat in their DNA. If one of them had it, there would have been a 415 bp fragment, and an 715 bp fragment. If both of them had it, there would only be a fragment at 715 bp. In my case, I only had a fragment at 415 bp, so neither of my parents has the Alu repeat.
Conclusion:
The lab was really about learning how to use lab equipment. We got to practice using laboratory materials such as centrifuges, microfuge tubes, and micro pipets. Overall, this was a great way to learn about safe lab practices, proper use of equipment (balancing the centrifuge), and PCR.