Purpose: To make a positive/negative control and DNA sample using V4 primers and COX1 primers

Materials

• TAE stock
• D.I. water
• Gel chamber
• Microwave
• COX1 primer
• V4 primer
• Environmental DNA sample
• Paramecium control sample
• Ethidium bromide

Procedure:

A. Primer solutions

1. Make 25ul reaction volume with PCR COX1 primers. In 6 separate centrifuge tubes, add 12.5 ul of 2X Master mix. To the negative control, add 11.88ul of H2O. To the positive control add 1 ul of the Paramecium control sample and 10.88ul of H2O. To the eDNA tube, add 1ul of the eDNA soil sample and 10.88ul of H2O.
2. To 3 tubes, add 0.63ul of COX1 primers and to the other 3 tubes, add 0.63ul of V4 primers. Each tube should be a 20uM stock.

B. Gel Electrophoresis

1. Make 1xTAE in 1L Erlenmeyer flask. Add 10mL of 10xTAE and 90mL of D.I. water
2. To make a 1.8% agarose gel, add 35mL of the 1xTAE to 0.63g of agarose in small Erlenmeyer flask
3. Cover flask lightly with weighing paper and loose-fitting cap
4. Heat until solution is clear and small bubbles come off the bottom when gently swirled
5. Allow to cool until the flask is comfortable to hold (5-6 minutes)
6. Add 2ul of ethidium bromide, swirl gently
7. Set up gel electrophoresis box, making sure the ends are sealed. Pour agarose gel smoothly into prepared mold, with as few bubbles as possible.
8. Insert the comb with its back towards the nearest edge. Allow to solidify for at least 25-30 minutes. Cover gel with 1xTAE stock so that it will not dry out. Carefully remove comb and store in the refrigerator.

Results:

Making the solutions for the PCR reactions and setting up our gel electrophoresis chamber went smoothly. Next week we will be adding our PCR solutions to the gel in order to perform gel electrophoresis. We are hoping this time there will be DNA present in our gels. There was significantly more pure DNA present in the groups that used the PowerSoil protocol rather than the Chelex protocol, but both produced sufficient DNA to proceed with our protocol.