During this lab we conducted a modified version of Chelex DNA extraction, prepared agarose gel for gel electrophoresis, and began PCR reactions with our newly extracted environmental DNA, a positive control with DNA taken from a paramecium culture, and a negative control with the DNA replaced with D.I. water.

Procedure:

Chelex DNA Extraction

(Each member of the group performs all steps with their own sample.)

1. Extract ciliates from a non flooded plate by taking a 1.5 ml sample.
2. Centrifuging it at 6000xg for 5 minutes.
3. Remove the supernatant.
4. Add 1.5 ml more to the sample and repeat steps 2 and 3.
5. Add 200 µL 5% Chelex to the sample (it is necessary to cut the end of of the pipette tip in order to extract Chelex).
6. Vortex for 1 minute.
7. Add 15 µl of proteinase K.
8. Incubate for 30 minutes in a 56°C water bath or heat block.
9. Boil for 8 minutes in 100°C heat block.
10. Vortex for 1 minute.
11. centrifuge at 16000xg for 3 minutes.

PCR

1. Each member transfer 100 µl of their supernatant to one clean microcentrifuge tube
2. In 3 small tubes transfer all of the following components excluding the DNA for the environmental sample, this will be done after step 3
   

   Negative Control	Positive Control	eDNA
   DNA template/ µL	0	1	1
   2X Master mix/ µL	12.5	12.5	12.5
   Stock V4 Primers (final concentration 0.5 µM)/ µL	1.25	1.25	1.25
   Water/ µL	11.25	10.25	10.25

   (in our experiment an error was made, and 11.25 µL of water was added to each tube)

3. Run a Nanodrop analysis of the environmental DNA sample.
   

   The indicated reading was from my groups Nanodrop analysis

4. Either concentrate, or dilute your DNA sample until it is approximately 50 ng/µL.
5. Transfer 1µL of your environmental DNA sample into the environmental DNA tube.
6. Place all three tubes in a thermocycler.

The indicated tubes are my groups samples

Agarose Gel Preparation

• Add  0.6g of agarose to an Erlenmeyer flask.
• Dilute with 40 mL of 1xTAE, in order to make a 1.8% agarose solution.
• Cover the flask with weighing paper and a loose fitting lid.
• Swirl the solution to mix
• Heat the erlenmeyer flask in a microwave for 1 minute and 20 seconds on power 7
• Cool the gel for 5 minutes in a water bath
• Add 2 uL of ethidium bromide
• Swirl the solution to mix
• Assemble gel mold
• Pour the gel into the mold, allow it to set
• Label the mold and store

Conclusion:

All this was a lot to do with our time constraints, and my group felt like we rushed it a bit.  However, we were able to finish with minimal extra time.  I did enjoy getting to go back and do some of the things we have done before.  That way all the procedures are more firmly cemented in my mind, so that I should be able to perform them even more efficiently in the future if its required.