Purpose:

The purpose of this lab was to prepare positive and negative control samples and an eDNA soil sample for PCR amplification by adding 2X Master Mix, DNA template (only to the positive control and soil sample), water, and the primer COX1. The PCR Amplification process includes denaturation, annealing, and elongation. In addition, we also prepared the gel needed for gel electrophoresis.

Procedures:

1. Sterilize the work environment by cleaning the table with bleach.
2. Add 2X Master Mix, DNA template, water, and primer to the designated tubes so that the volume of each tube is 25uL. (Note: See the table in “data/observations” for specific quantities.)
3. Add 40mL 1xTAE to 0.6g agarose in a small Erlenmeyer flask.
4. Cover lightly with weighing paper/Kimwipe and loose-fitting cap (Note: Do NOT tighten tightly close the cap otherwise an explosion might occur.)
5. Heat until solution is clear and small bubbles come off the bottom when gently swirled.
6. Allow to cool until the flask is comfortable to hold (5-6 minutes).
7. Have your TA add 2uL ethidium bromide, swirl gently.
8. Set up gel electrophoresis box, making sure the open ends are sealed.
9. Pour agarose gel smoothly into prepared mold, with as few bubbles as possible. Insert the comb with its back towards the nearest edge. Allow it to solidify for at least 25-30 minutes.
10. Cover gel with prepared 1xTAE buffer solution so that it will not dry out.
11. Carefully remove comb and turn gel so that the wells are furthest away from positive electrode (think “run to red–you want the negative DNA to run towards the red positive electrode.)
12. Use a micropipetter, add 5uL of the ladder and 10uL of each pCR product + loading buffer. If the loading buffer is not included in the Taq polymerase used in the PCR, add 5uL loading buffer to the PCR product and mix thoroughly before transferring to the gel.
13. After you have loaded your sample, place the lid on your box and turn on the power supply to approximately 100 volts. Allow to run for 30 minutes or more, allowing the loading dye to run approximately halfway across the gel before turning off the power.
14. Image with UV light.

Data/Observations:

Calculation determining the amount of agarose:

X g agarose / 40mL buffer = 0.015

X = (40mL)(0.015)

= 0.6 g agarose

Each tube (from step 2 of the procedures) is labeled with a “6” on the top and a “+”, “-“,  or “s” on the side. Our gel rack is labeled “6 LBP”

Conclusion:

This lab was great exposure to the PCR amplification process and was the next step in allowing us to classify ciliates based on their genetics. I thought it was really cool that we we were able to make our own gel and that this protocol was fairly hands on. I am excited to run the gel electrophoresis next week and to finally be able to analyze the results of our organisms.