Lab 3: Ciliate Isolation and DNA Extraction Protocols 01/25/18
Purpose/Objective
The purpose of today’s lab was to design a protocol for metabarcoding of soil ciliates to explore ciliate biodiversity. We also learned how to isolate ciliates using Ludox gradient centrifugation
Procedure
- Overall Workflow
- Cell Isolation: Ciliates (and other cells) are separated from soil
- DNA Extraction: Use methods that results in “clean” DNA
- PCR/Gel Electrophoresis Validation: Use of primers for sequencing
- Metabarcoding (High Throughput Amplicon Sequencing)
- Analysis of Sequencing Data
Today we performed the following Ludox gradient centrifugation protocol to isolate the ciliates, and begin extracting the DNA
- Add 8-mL of Ludox HS 40 to a 15-mL conical tube
- Quickly add (inject) 2-mL of liquid from the soil samples in the jars into the Ludox using a p100. (Cut the tip to avoid getting soil stuck. Avoid grass and sticks)
- Carefully add (layer) 2-mL of distilled water to the top of the Ludox mixture. Don’t mix.
- Centrifuge in a swinging bucket rotor for 15 min at 4300 x g. (Make sure your tubes are equal mass before loading the centrifuge. Add water to balance)
- Carefully remove the tube and use a pipette to remove the cell layer (visible uder the water layer)
- Transfer to a clean microfuge tube. (If more than 1-mL, use 2 tubes.
- Transfer 3 10-mL drops to a concavity slide and count the cells using 100x magnification. (Record your observations and if possible, estimate cells/mL)
Results
Our centrifuge tube ended up weighing approximately 21.4 grams. The image below is a picture I took with my phone, of my partner Ben’s slide.
Conclusion
I heard from a friend that this lab was going to be fun, and he was right! The only bummer was that my microscope’s rheostat was broken so the light was far too bright for my eyes to see my slide, but I was still able to look at my lab partners samples under their microscopes! This lab was really interesting because not only did I get more hands on experience with ciliates, I also got to know my classmates from groups 5 and 6 better, along with my TA Will.