Purpose:

The purpose of this lab was to learn how to do Ludox Centrifugation and ultimately isolate cells from our soil sample. We did this in order to prepare for DNA isolation that we will be preforming next class.

The protocol we came up with for the day was:

Soil Sample- collected soil sample

Ludox Centrifugation- we created our centrifugation with the ludox

Cell Isolation- we took the one layer of cells and isolated it in its own tube to store for later

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.

Data:

This is the ciliate I found in the drops I looked at. My tube weighed 21.3g.

Conclusion:

This lab helped us to prepare for DNA extraction which we will perform in the next lab. We will later use PCR and Metabarcoding in order to figure out the species we have found. We were successful in isolating cells and hopefully will be able to analyze the DNA next class.

Storage:

Put our centrifuge tube in the freezer labeled ams.