Task, Rationale, and Purpose: Lab week 7 introduced polymerase chain reaction (PCR) along with the construction of an agarose gel electrophoresis setup. The purpose of this lab is to be able to create multiple copies of a specific sequence from the DNA that the lab isolated from last week’s protocol. Secondly, the procedure calls for the set up for a gel electrophoresis experiment. The task or rationale for this week’s lab is to gain firsthand experience in PCR and gel electrophoresis experiments. For future labs and research, this knowledge is necessary for almost all fields of science.

Procedure:

1. Ensure that the lab table is bleached and all participants are wearing gloves.
2. Obtain three microfuge tubes containing 12.5 microliters of 2X master mix.
3. Label one tube “+”, the second tube “-“, and the third tube “S”.
4. Using a micropipette p10, add 1 microliter of Cox1 and Cox2 primer to each tube.
5. Using a micropipette p10 with a new tip, add 5 microliters of the environmental DNA obtained from last week’s lab protocol into the S tube.
6. Using a micropipette p10 with a new tip, add 5 microliters of positive control DNA into the + tube.
7. Using a micropipette p20, add 11.5 microliters of DI water to the – tube.
8. Using a micropipette p20 with a new tip, add 6.5 microliters of DI water to the + tube.
9. Using a micropipette p20 with a new tip, add 6.5 microliters of DI water to the S tube.
10. Place the tubes in the class tube rack and record their location on the rack and designate them with a group number and group initials.
11. Begin the process of PCR by placing the tubes in a thermal cycler. Heat the tubes to 94 degrees Celsius for 4 minutes.
12. Undergo 5 cycles of denaturation at 94 degrees Celcius for 30 seconds, annealing at 45 degrees Celsius for 1 minute, and extension at 72 degrees Celsius for 105 seconds.
13. Undergo 35 cycles of denaturation at 94 degrees Celcius for 30 seconds, annealing at 55 degrees Celsius for 1 minute, and extension at 72 degrees Celsius for 105 seconds.
14. Continue to keep the thermal cycler at 72 degrees Celsius for 10 minutes.
15. Store the tubes at 4 degrees Celsius.
16. Obtain 40 mL of 1xTAE (Tris Acetate EDTA) and add 0.6 grams of agarose to it in an Erlenmeyer flask.
17. Cover the flask with a Kimwipe and a cap. Ensure the cap is loose.
18. Heat the solution until bubbles form on the bottom.
19. Cool the gel for 5-6 minutes.
20. Add 2 microliters of ethidium bromide and swirl.
21. Prepare a gel electrophoresis box.
22. Pour the cool agarose gel into the mold and use a micropipette to pop any bubbles that form.
23. Insert the comb into the furthest holder to the back of the box.
24. Add the prepared 1xTAE buffer solution and remove the comb.
25. Position the gel with the wells farthest from the positive electrode.
26. Add 5 microliters of the ladder and 10 microliters of each PCR product with loading buffer into the wells. Ensure that each PCR product is in different wells.
27. Put the lid on the box and turn the power on supplying it with 100 volts for 30 minutes at the minimum.
28. Image the samples with a UV light.

Data/Results:

Tube rack Information: F1, F2, and F3; Group 8; KSA

Agarose gel electrophoresis box label: Room – A127, Teacher – Dr. Adair, Name – KSA

Gel electrophoresis results to be determined in next week’s lab

Conclusion: The procedure was followed and completed successfully. There are a couple of errors, however, that the group had trouble with. Correct measurements were skeptical and one of the tubes was dropped. Additionally, an entire tray of micropipette tips was dropped. Regardless of the errors, the rest of the experiment continued flawlessly. Expectations for the electrophoresis results are low due to the amount of contamination in the original environmental DNA sample. For future studies, the protocol should be revised and enacted in a more professional and timely manner. For example, the addition of the materials into the microfuge tubes should be done in a fume hood to avoid foreign DNA contamination. Furthermore, hairnets and extra safety precautions should be enacted. Ultimately, the continuation of the gel electrophoresis experiment will commence next week.