Date: 3/16/18

Title: Chelex, MoBio

Purpose: Our goal is to extract DNA from cells to identify ciliates and learn more about the diversity of ciliates.

Materials:

(MoBio)

• Microcentrifuge (10,000 x g)
  Pipettors (50 μl – 500 μl)
  Vortex-Genie® 2 Vortex
  Vortex Adapter, powerBead tubes, ethanol

(Chelex)

• Chelex Beads, non-flooded plate, vortex, centrifuge

Procedure:

(MoBio)

1. add 0.2-0.3 grams of soil sample to powerbead tube
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  vortex
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter
tube holder, Vortex at maximum speed for 10
minutes
6.  Centrifuge tubes at 10,000 x g for 30 seconds
7. Transfer the supernatant to a clean 2 ml Collection Tube
8. Add 250 μl of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5
minutes
9. Centrifuge the tubes  for 1 minute at 10,000 x g
10.  transfer up to 600 μl of supernatant to a clean 2 ml
Collection Tube
11. Add 200 μl of Solution C3 and vortex, Incubate at 4°C for 5 minutes
12. Centrifuge the tubes for 1 minute at 10,000 x g
13. Transfer up to 750 μl of supernatant to a clean 2 ml Collection Tube
14. Add 1.2 ml of Solution C4 to the supernatant, vortex for 5
seconds
15. Load 675 μl onto a Spin Filter and centrifuge at 10,000
x g for 1 minute, 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, Load the remaining supernatant onto the Spin
Filter and centrifuge at 10,000 x g for 1 minute
16. Add 500 μl of Solution C5 and centrifuge for 30
seconds at 10,000 x g
17. Discard the flow through from the 2 ml Collection Tube (avoid traces of ethanol)
20. Add 100 μl of Solution C6 to the center of the white filter membrane
21. Centrifuge for 30 seconds at 10,000 x g
22. Discard the Spin Filter, the DNA in the tube is ready for any downstream
application

Chelex

1. Transfer 1.5 ml of liquid (from non-flooded plate) to a microcentrifuge tube
2. label tube with your initials and section
3. Centrifuge @6000xg for 5 minutes, discard supernatant
4. repeat procedure 2-3 times to concentrate the cells in the liquid into a pellet
5. 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, vortex for 1 minute/ use large-bore micropipette tip
   2. Add 15 µl of proteinase K
6. Incubate for 30 minutes in 56^oC water bath or heat block
7. Boil for 8 minutes in 100^oC water bath or heat block
8. Vortex for 1 minute
9. Centrifuge @ 16,000xg for 3 minutes to pellet cellular debris and Chelex beads
10. Transfer supernatant with DNA in solution to clean microcentrifuge tube
11. Label top and side of microcentrifuge tube with ID!!!

Data:

Our group completed the MoBio protocol. The wieght of the dry soil was 0.2g.

Mistakes: Issues with pipetting can be a source of error and it results in the layers of the tube being less distinct. Another issue would be accidentally pipetting the pellet.

Location: Lab room, labeled PSCLK

Conclusion:

We did not complete the procedure so there are no conclusive results to report.

next step: We will be creating the electrophoresis gel. This is how we will test to see if there is DNA. If the test comes back postitive, then we can begin to classify the ciliates based on the DNA that was read.