Purpose

The purpose of this lab was to perform two sets of extraction methods and two sets of primers. Our hope was to have better results than we did from the Ludox protocol. Each group, performing either the Chelex or PowerSoil protocol, tested their extracted DNA and compared it to their positive and negative control groups. Two different types of primers were used: COX1 and V4 primers. The reason for this was to test the success of the DNA extraction methods we performed in the previous lab. After the reactions were set up, each group prepared the agarose gels so we can continue the analysis of DNA using gel electrophoresis. The gel was needed to be able to determine how successful our efforts have been during these last few lab sessions.

Procedure
Reaction Set-Up

1. Label tubes 1-6 and keep track of which tubes contain a specific solution 2. Add 0.63 mL of the 20x stock COX1 primer solution to tubes 1-3 that contain the master mix

3. Add 0.63mL of the 20x stock V4 primer solution to the tubes 4-6 that contain the master mix

4. Add 11.87 mL of water to tubes 1-4

A. This will be the negative control

5. Add 10.87 mL of water to tubes 2-3 and 5-6

A. Tubes 2 and 5 will be the positive control

a. Add 1ul of the positive control solution to these tubes

6. Add 1ul of eDNA collected from Powersoil or Chelex protocol to tubes 3 and 6

7. Store the tubes and record their location
1.8% Agarose TAE Gel Set-Up

1. Pour 90mL of D.I. water into flask

2. Add 10mL of 10x TAE

3. Add 0.63 grams of agarose to 35mL of the prepared 1X TAE solution from step 1

4. Mix and heat in microwave followed by some cooling time

5. Pour gel in the gel tray and allow it to cool for the remainder of lab

6. Label the gel tray and store the tray where instructed

Data and Observations

• Our six tubes were labeled with number 1-6 followed by our group members initials “TS”

• Tubes 1-3 contain COX1 primers

• Tubes 4-6 contain V4 primer

• Gel tray labeled: 21-TCa

• Calculation to find amount of agarose added: (0.018)(35) = 0.63 grams of agarose

• Must round to the nearest hundredth due to pipettes not being able to draw to the thousandths place

Conclusion and Future Steps 

At the end of this lab, we had successfully set up a reaction that will determine which protocol and which primers are the most effective. The final step is the electrophoresis gel that will allow us to examine any DNA and the sequences amplified. The electrophoresis will take place in the following lab. Our lab group hopes that these protocols and primers are successful and produce positive results that we can replicate in the future.