Purpose

• Describe the main points from the review of literature
• Explain how ciliates may be isolated from a soil sample using Ludox centrifugation
• Describe the polymerase chain reaction and how it is important in metabarcoding
• Cell separation
• DNA amplification

Materials

• Ludox HS 40
• Conical tube
• Soil sample
• Swinging bucket rotor
• Distilled water
• Microscope

Procedure

1.Add 8ml Ludox HS 40 to a 15 ml conical tube.

2.Quickly add (inject) 2 ml of liquid from the soil samples in the jars into the Ludox using a p1000. Avoid grass and sticks…that are low density.

3.Carefully add (layer) 2 ml of distilled water to the top of the Ludox mixture. Don’t mix.

4.Centrifuge in a swinging bucket rotor for 15 min at 4300 x g.

5.Carefully remove the tube and use a pipette to remove 3-5 ml layer of cells (under the water layer)

6.Transfer to a clean 15 ml comical tube.

7.Dilute with buffer to 10 ml and spin at 4300 x g for 10 minutes to wash and pellet the cells.

8.Remove (decant) the supernatant and store the pellet in the freezer.

Results

We were able to extract the organisms by Ludox centrifugation with a pipette. Ciliates were visible under a compound microscope however, none were mobile.

Conclusion

Our ciliate extractions will sit in the freezer for about a week and then we will proceed with our protocol.