Lab 4: 2/1/18

Purpose:

The purpose of todays lab was to work as a class to discuss the issues we had from last weeks protocol and collaboratively fix each issue. After creating a possible protocol, we considered positive and negative control groups. We then did the protocol we created. 

Procedure:

• Part A

1. As a group, reflect on the issues we had with the previous protocol from last week and brainstorm possible ways to improve those issues.
2. Think of  ways in which we can possibly incorporate a positive control.

• Part B

1. Weigh out 5 grams of soil (screened of debris)
2. Add 7 mL water; mix either by hand or with vortex  for 5-10 mins
3. Transfer 1800 microliters to tube; add 200 microliters of 25% Glutarol.
4. Vortex briefly to mix well.
5. Inject 2 mL of fixed sample to Ludox tube by placing P1000 tip at 6mL mark.
6. Carefully layer 2 mL of colored water on top of Ludox.
7. Centrifuge 4300 xg for 15 mins.
8. Remove 2 mL of cells (organic layer).
9. Count five 2 microliter drops from sample and get class average.
10. Centrifuge the 2 mL (or two 1.5 mL) tubes 15,000g for 1 minute. Spin with hinge (or mark) on outside, so you know pellet side.
11. Remove 2,000 microliters of supernatant with P1000. If you leave a small layer of liquid in tube (5-10 microliters), it is okay. Freeze for DNA extraction.

Data:

• Part A

Some of the issues we had last week and are wanting to fix with this protocol and some solutions to the problems are:

1.Starting all samples off with the same amount of soil.

• 5g of soil.

2. Get all debris out.

• Wire net to screen debris.

3. More Ludox tubes.

• separate organic layer into several tubes for each individual person.

4. Make cell layer more obvious.

5. Cells lost during pipetting

• Rinse pipette
• Dispense slowly

• Part B

We were able to improve the protocol by all using the same weighted sample. As a class we decided 5g of soil was a good starting point. We added on a positive control to our protocol by doing the exact same procedure with Tetrahymena. In doing this, we will be able to compare our results with our grass sample to that of the Tetrahymena sample, to ensure were getting accurate results. The protocol for the Tetrahymena sample will be the same as the sample:

1. weigh out 5g of autoclaved soil.
2. Add 2 mL of Tetrahymena culture (about 2000 cells)
3. Add 5 mL of water. (not 7 because the Tetrahymena will already be in 2mL, this will keep our weights the same.)

• Part C

Our soil sample weight was 5g.

The soil, ludox and sample together weighed 21g.

Conclusion:

Todays lab was a lot of fun since we designed our protocol as a class. We really put something together as a team and I think it’s going to work out successfully. I really liked that we brought up the issues we had last lab and worked to think of ways to fix those problems. Although it didn’t work efficiently, I’m glad I was able to contribute to the idea of screening the debris out of our sample. I also really liked how were going back to working with Tetrahymena as our positive control.

Storage:

My groups conical tube is stored in a test tube rack for Dr. Adair to centrifugate; it is labeled “LAK”.

Future Steps: As a group or one of us from our group will come to open lab tomorrow to continue working on our protocol. At open lab we will isolate cells from the organic layer and store them away, probably in a cooler. I really hope we start getting into the DNA sequencing soon; I imagine that’s the best part of this lab.