Purpose:

The purpose of this lab was to extract the DNA from the given soil sample and start a new experiment as our last one failed.

Procedure: 

Chelex Procedure:

• Transfer 300-500uL of dense ciliate culture to a microcentrifuge tube. This culture should be taken from one of the non-flooded plates available.
• Label the tube with the group number and initials.
• Centrifuge at 6000xg for 5 minutes and discard the supernatant.
• Weigh 0.5g of Chelex and transfer to a 15mL conical tube. Add 10mL of D.I. water as well.
• Add 200uL of 5% Chelex to pellet and vortex for 1 minute.
• Using a micropipette, add 15uL of proteinase K. This step may require the cutting of the tip of a 1000uL micropipette.
• Incubate for 30 minutes in a 56 degrees Celsius water bath.
• Boil for 8 minutes in a 100 degree Celsius water bath.
• Vortex for 1 minute.
• Centrifuge at 16,000xg for 3 minutes to pellet cellular debris and Chelex beads.
• Transfer the supernatant with the DNA in the solution to a clean microcentrifuge tube. Be sure to not transfer the Chelex beads.
• Label the top and side of the microcentrigue tube with you ID.

Powersoil Procedure:

• Weigh our 0.3 grams of soil sample and add to the PowerBead tubes provided.
• Gently vortex to mix.
• Check solution C1. If solution C1 is precipitated, heat the solution to 60 degrees Celsius until the precipitate has dissolved before use.
• Add 60uL of the solution C1 and invert several times.
• Secure the PowerBead tubes horizontally using the MO BIO Vortex Adapter tube holder. Vortex at the maximum speed for 10 minutes.
• Centrifuge tubes at 10,000xg for 30 seconds at room temperature.
• Transfer the supernatant to a clean 2mL collection tube.
• Add 250uL of solution C2 and vortex for 5 seconds. Incubate at 4 degrees Celsius for 5 minutes.
• Centrifuge the tubes at room temperature for 1 minute at 10,000xg.
• Avoiding the pellet, transfer up to 600uL of supernatant to a clean 2mL collection tube.
• Add 200uL of solution C3 and vortex briefly. Incubate at 4 degrees Celsius for 5 mixtures.
• Centrifuge the tubes at room temperature for 1 minute at 10,000xg.
• Transfer up to 750uL of supernatant to a clean 2mL collection tube.
• Shake to mix solution C4 before use. Add 1.2mL of solution C4 to the supernatant and vortex for 5 seconds.
• Load 675uL onto a spin filter and centrifuge at 10,000xg for 1 minute at room temperature. Discard the flow through and add an additional 675uL of supernatant to the spin filter and centrifuge at 10,000xg for 1 minute at room temperature. Load the remaining supertanat on the spin filter and centrifuge at 10,000xg for 1 minute at room temperature.
• Add 500uL of solution C5 and centrifuge at room temperature for 30 seconds at 10,000xg.
• Discard the flow through from the 2mL collection tube.
• Centrifuge at room temperature for 1 minutes at 10,000xg.
• Carefully place spin filter in a clean 2mL collection tube.
• Add 100uL of solution C6 to the center of the white filter membrane.
• Centrifuge at room temperature for 30 seconds at 10,000xg.
• Discard the spin filter.
• Store the DNA in a -20 to -80 degrees Celsius environment.

Results:

We did not obtain any results as we were only performing the procedure. 

Conclusion:

Our group performed the Powersoil procedure and we think it went very well. There was no observable signs of error. Our other group performed the Chelex procedure which was a little different than ours and is said to be less accurate. Next week, we will perform PCR to extract the DNA therefore further preparing it for analyzation. The purpose for putting in 0.3 grams was to account for any loss of sample when it was transferred from one tube to another. I hope this time around we will be able to see positive results and that the change in the procedure will allow us to see the diversity in the soil sample.