During this class time we began the process of two new DNA extraction methods.  PowerSoil and Chelex.  Each table began the extraction using one sample for each method.  A control using a paramecium culture was made for each Chelex tube, and one control was made for all the PowerSoil samples.  This discrepancy is due to the time restraints of our experiment, and the time consuming nature of using the PowerSoil method.

Procedures:

Chelex:

Modified Chelex Extraction Protocol

Adapted from Stüder-Kypke, 2001

1. Transfer 300-500 µl of dense ciliate culture (20 or more individuals) to a microcentrifuge tube. If you do not have your own culture, perform a replicate from another student’s culture.
   1. Make sure to record which ciliate culture you are extracting from
2. Label your tube with your initials and section and record all the information about the sample in your notebook.
3. Centrifuge @6000xg for 5 minutes, discard supernatant

For Chelex extraction from non-flooded plates, remove 1.5 ml of liquid from the non-flooded plate.  Spin at 6000xg for 5 minutes and remove the supernatant.  Repeat this procedure 2-3 times to concentrate the cells in the liquid into a pellet.  Avoid pipetting the soil.

Chelex is a chelating resin that chelates metal ions that are required for enzyme activity.  This reduces the amount of enzymatic activity of the nucleases and therefor protects DNA integrity.

4. Weigh 0.5 g Chelex and transfer to a 15 ml conical tube. Add di water to 10 ml.
   1. Add 200µL 5% Chelex to pellet, and vortex for 1 minute.
   2. For this step, use large-bore micropipette tips or simply cut off the tip of a 1000µL micropipette tip
   3. Add 15 µl of proteinase K.
5. Incubate for 30 minutes in 56^oC water bath or heat block: This will break open the cells and denature some proteins.
6. Boil for 8 minutes in 100^oC water bath or heat block.
7. Vortex for 1 minute
8. Centrifuge @ 16,000xg for 3 minutes to pellet cellular debris and Chelex beads
9. Transfer supernatant with DNA in solution to clean microcentrifuge tube, being careful not to transfer Chelex beads
10. Carefully label top and side of microcentrifuge tube with your ID that you will be able to identify.

PowerSoil:

Detailed User Protocol provided by MO BIO Laboratories Inc.

1. To the PowerBead Tubes provided, add 0.25 grams of soil sample (this was rounded either to .2 or .3 due to scale limitations)
2. Gently vortex to mix.
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60°C until
   the precipitate has dissolved before use.
4. Add 60 μl of Solution C1 and invert several times or vortex briefly.
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter
   tube holder for the vortex (MO BIO Catalog# 13000-V1-24) or secure tubes
   horizontally on a flat-bed vortex pad with tape. Vortex at maximum speed for 10
   minutes.
6. Make sure the PowerBead Tubes rotate freely in your centrifuge without
   rubbing. Centrifuge tubes at 10,000 x g for 30 seconds at room temperature.
   CAUTION: Be sure not to exceed 10,000 x g or tubes may break.
7. Transfer the supernatant to a clean 2 ml Collection Tube (provided).
8. Add 250 μl of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5
   minutes.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer up to 600 μl of supernatant to a clean 2 ml
    Collection Tube (provided).
11. Add 200 μl of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Transfer up to 750 μl of supernatant to a clean 2 ml Collection Tube
    (provided).
14. Shake to mix Solution C4 before use. Add 1.2 ml of Solution C4 to the
    supernatant (be careful solution doesn’t exceed rim of tube) and vortex for 5
    seconds.
15. Load approximately 675 μl onto a Spin Filter and centrifuge at 10,000
    x g for 1 minute at room temperature. Discard the flow through and add an
    additional 675 μl of supernatant to the Spin Filter and centrifuge at 10,000 x g
    for 1 minute at room temperature. Load the remaining supernatant onto the Spin
    Filter and centrifuge at 10,000 x g for 1 minute at room temperature.
16. Add 500 μl of Solution C5 and centrifuge at room temperature for 30
    seconds at 10,000 x g.
17. Discard the flow through from the 2 ml Collection Tube.
18. Centrifuge at room temperature for 1 minute at 10,000 x g.
19. Carefully place Spin Filter in a clean 2 ml Collection Tube (provided).
    Avoid splashing any Solution C5 onto the Spin Filter.
20.  Add 100 μl of Solution C6 to the center of the white filter membrane.
21. Centrifuge at room temperature for 30 seconds at 10,000 x g.
22. Discard the Spin Filter. The DNA in the tube is now ready for any downstream
    application. No further steps are required.

My group (22-5) completed the Chelex: extraction method for our table.

Control (left) and environmental (right) samples before centrifugation

Our Environmental sample in the 56°C bath