Objective

• Practice DNA purification on the eDNA we extracted from our soil samples
• Set up gel electrophoresis tests
• Begin setting up our methods for our research

Procedure

Chelex Procedure:

1. Incubate sample for 30 minutes at 56°C on a heat block
2. Broil for 8 minutes at 100°C on the heat block
3. Vortex for 1 minute
4. Centrifuge at 16000 xg for 3 minutes
5. Transfer the supernatant to a clean tube and discard the pellet
6. Store the tube in a freezer box

Agarose Gel Procedure:

1. Make one liter of 1xTAE solution by adding 4mL of 10x TAE stock solution to 36 mL of DI water and add 0.4 grams of agarose.
2. Swirl gently in a flask
3. Heat the solution in the microwave for 2 minutes
4. Swirl gently again
5. Let it cool for a couple of minutes
6. Add 0.002 micro-liters of Ehthidium Bromide to the solution to make it a 0.5 ug/mL concentration in the solution. Swirl gently
7. Set up the gel electrophoresis box with rubber bumpers and then add the comb to make the wedges in the gel
8. Add the solution to the box
9. Allow the solution to solidify ~ 30 minutes
10. After the solution solidifies, add a small amount of 1xTAE buffer over the gel
11. Place the box in a plastic bag and store in the fridge

Conclusion

We were able to make a gel mold for our gel electrophoresis test and continued upon the Chelex protocal to finish extracting our DNA. The procedure for both protocols went smoothly. We also were able to check the soil sample cultures. These cultures were the same ones that we cultivated a couple of weeks ago when we were still trying to isolate ciliates from the samples.

Future Steps

The agarose gel we created was put in a bag and stored in the fridge. The DNA samples were also put in the freezer box. Next week, we will be continuing and go off of what we did this week.