3/15/18

Today in lab our group did the Powersoil procedure and the other group did the Chelex procedure.  In both procedures the goal was to extract DNA.  We did 2 different protocols to test which one yields better results.  We are tried different protocols because no DNA appeared in the gel electrophoresis

Procedures

Powersoil

1. Obtain a PowerBead Tube and add 0.25 grams of dry soil
2. Gentry vortex to mix
3. Add 60 µL of Solution C1 and vortex briefly
4. Secure the PowerBead Tubes horizontally using the MO BIO Vortex and vortex for 10 minutes at maximum speed
5. Centrifuge the tube at 10,000 g for 30 seconds
6. Transfer all the supernatant from the PowerBead Tube to a clean 2 mL collection tube
7. Add 250 µL of Solution C2 and vortex for 5 seconds
8. Incubate for 5 minutes at 4°C in the refrigerator
9. Centrifuge the tube at 10,000 g for 1 minute
10. Transfer up to 600 µL of the supernatant to a clean 2 mL collection tube; make sure to avoid the pellet when doing so
11. Add 200 µL of Solution C3 and vortex briefly
12. Incubate for 5 minutes at 4°C in the refrigerator
13. Centrifuge the tube at 10,000 g for 1 minute
14. Transfer up to 750 µL of the supernatant to a clean 2 mL collection tube
15. Add 1.2 mL of Solution C4 to the supernatant and vortex for 5 seconds
16. Obtain a Spin Filter
17. Load 675 µL of the solution into the spin filter and centrifuge at 10,000 g for 1 minute
18. Repeat that step until all the solution is gone, but keep the spin filter
19. Add 500 µL of Solution C5 to the spin filter
20. Centrifuge at 10,000 g for 30 seconds
21. Discard the flow through from the 2 mL collection tube
22. Centrifuge at 10,000 g for 1 minute to dry and remove the remaining Solution C5
23. Carefully remove the spin filter and make sure no flow through splashes it
24. Place the spin filter into a clean collection tube
25. Add 100 µL of Solution C6 to the center of the spin filter membrane
26. Centrifuge at 10,000 g for 30 seconds
27. Discard the SPIN FILTER and keep the filtrate
28. Label and store DNA (-20°C to -80°C)

Chelex

1. Transfer 300-500 µL of dense ciliate culture (Paramecium culture) to a micro centrifuge tube
2. Label the tube
3. Centrifuge at 6,000 g for 5 minutes
4. Discard the supernatant
5. Weigh 0.5g Chelex and transfer to a 15 mL conical tube
6. Add 10 mL of DI water
7. Add 200 µL of 5% Chelex to the pellet
8. Vortex for 1 minute
9. Add 15 µL of proteinase K
10. Incubate for 30 minutes in 56^oC heat block
11. Boil for 8 minutes in 100^oC heat block
12. Vortex for 1 minute
13. Centrifuge at 16,ooo g for 3 minutes
14. Transfer supernatant with DNA in solution to clean micro centrifuge tube
15. Label and store DNA

Observations

0.3 g of soil

Thoughts

On the last step of the powersoil procedure, our group accidentally disposed of the liquid instead of the filter and ended up throwing out our DNA. [I know we messed up pretty bad 🙁  ] So Dr. Adair told us to run 100 uL more of the C6 through the filter again to hopefully get some DNA out but our DNA concentration will probably be much lower than the other groups if we even have any DNA.  We labeled our tube 8.