Purpose: to perform the new Chelex and PowerSoil DNA extraction protocols.

Procedure:

Chelex:

1. Remove 1.5 mL of liquid from a pre-made non-flooded plate
2. Spin at 6000xg for 5 minutes and remove the supernatant
3. Repeat step two 3 times
4. Weigh 0.5 g of Chelex and transfer into a 15 mL conical tube. Add D.I. water up to the 10 mL mark
5. Add 200 uL of 5% Chelex to the pellet and vortex for 1 minute, then add 15 uL of proteinase K
6. Incubate for 30 minutes in a 56 degree Celsius heat block
7. Boil for 8 minutes in a 100 degree Celsius heat block and vortex for 1 minute
8. Centrifuge at 16,000xg for 3 minutes
9. Transfer supernatant to clean microcentrifuge tube
10. Label the top of the tube and store in freezer
11. Simultaneously conduct this procedure using 500 uL of Paramecium culture in a different tube to serve as the positive control

PowerSoil:

1. Obtain PowerBead tube and add 0.25 g of dry soil and vortex
2. Add 60 uL of Solution C1 and vortex
3. Use the MO BIO vortex to mix horizontally and vortex for 10 minutes at maximum speed
4. Centrifuge tube at 10,000xg for 30 seconds
5. Transfer supernatant into a clean 2 mL collection tube
6. Ad 250 uL of Solution C2 and vortex for 5 seconds
7. Incubate at 4 degrees Celsius in refrigerator
8. Centrifuge tube at 10,000xg for 5 seconds
9. Transfer ~600 uL of supernatant into a clean 2 mL collection tube
10. Add 200 uL of Solution C3 and vortex
11. Incubate in 4 degrees Celsius for 5 minutes
12. Centrifuge tube at 10,000xg for 1 minute
13. Transfer ~750 uL of supernatant into a clean 2 mL collection tube
14. Add 1.2 mL of Solution C4 to supernatant and vortex for 5 seconds
15. Obtain a spin filter and load 675 uL of solution into it
16. Centrifuge at 10,000xg for 1 minute; repeat until all solution is gone
17. Add 500 uL of Solution C5 to spin filter and centrifuge at 10,000xg for 30 seconds
18. Discard spin filter but keep the filtrate
19. Label and store in freezer

Results:

My group conducted the Chelex procedure. We followed the procedure as exactly as we could and successfully made our experimental tube; however, we forgot about the positive control tube until we were about halfway through with our experimental one. As a result, we were unable to complete the procedure for the positive control tube and only got through step 6. We shortened some of the times along the way by ~1 minute in an attempt to finish on time, but we will need to go into open lab to finish our positive control tube.

We labelled our tubes on the top with 2 MS WS CC and on the sides and stored the experimental one in the freezer. We left the positive control one in the heating block to finish up step 6, and Michael said he would take it out so we could come into open lab and finish the procedure tomorrow.

Next Step:

The next step will be to look at the solution using a nanodrop to see if there is any DNA in the samples. If there is, we can continue with PCR and gel electrophoresis; if there is not, we will need to look for an alternate procedure.