2/1/2018, Ludox gradient centrifugation protocol

Purpose: The purpose of this lab was to revise our Ludox protocol to make it more efficient to yield better results. We will be using the Ludox method to extract cells for DNA analysis.

Materials:

• Ludox
• Gluteraldehyde
• Grassland soil
• centrifuge

Procedure:

• Place 10 grams of soil and 20 grams of water in a baby food jar.
• Mix for 5 minutes and let settle for 1 minute.
• Add 900 µl (3 times) of soil juice into the Ludox at the 5 mL mark.
• Add 2 mL of food colored water to the top layer of the mixture.
• Weigh the tube on a scale (make sure that the weight matches the other group in your table!)
• Centrifuge on 4300 g for 15 minutes.
• Place in the refrigerator.
• Draw 2 mL of the organic matter from the tube (pull from the top).
• Take 5, 2 µl drops of the mixture in the tube and count the cells.
• Calculate the efficiency.
• Pellet out the cells in the microfuge tube by spinning at 1200 g for 1 minute.

Data: the weight of the Ludox tube was 23.1 grams

Results:

We were not able to complete the process so there are no results to analyze yet. Sources of error could come from not ensuring that the Ludox tubes at each table were the same weight. My groups Ludox tube did not match the weight of the other groups Ludox tube, so we had to add more water. This means that we added more water than the protocol initially called for.

Conclusion:

Since we have not finished the procedure, there is nothing to conclude yet. The next step would be to place our Ludox tubes in the centrifuge for 15 minutes, and then we will extract the cells for counting. We have also decided on a method to establish our control. We will add extra Tetrahymena to the same grassland soil sample we are using and repeat the Ludox protocol on this control group.

Where is it stored:

Our tube is labelled “CKL” on the side and it has our weight (23.1 g) listed on the cap. It is stored on a tube holder in the lab.