Purpose/Objective

The purpose of today’s lab was to perform DNA extraction using the EZNA Tissue DNA Kit with a protocol that was modified specifically for targeting ciliates.

Procedure

First we observed the cells from the last lab under a microscope and counted the number of cells in 200 μL of solution.

1. Spin tubes that were stored from last class for 5 minutes at 3000g
2. Remove the supernatant, taking care to not disturb the pellet
3. Add 1 mL of 1X PBS to resuspend
4. Spin tubes again for 5 minutes at 3000g
5. Remove the supernatant, taking care to not disturb the pellet
6. In a separate tube, add 20 uL of Iodine and 20 uL of cells
7. Place 5 2uL drops on a concavity slide and view under the microscope; count cells and record

We then proceeded with the EZNA Tissue DNA Kit Protocol

1. Prepare the cell suspension: Wash the cells by adding 200 uL of PBS and then vortex for a few minutes
2. Add 25 uL of OB Protease Solution and vortex to mix
3. Lysis: Add 220 uL of BL Buffer
4. Incubate tube at 70 degrees Celsius for 10 minutes in the heating block, then vortex
5. Binding: Add 220 uL of 100% ethanol and vortex to mix
6. Insert an HiBind DNA mini column into a 2 mL collection tube
7. Transfer the entire sample into the HiBind DNA mini column, including any precipitates that have formed
8. Centrifuge at maximum speed for 1 minute
9. Discard the filtrate and reuse the collection tube
10. Wash and dry: Add 500 uL of HBC Buffer to the column
11. Centrifuge at maximum speed for 30 seconds
12. After the HBC Buffer was, discard the filtrate and collection tube
13. Insert the HiBind DNA mini column into a new collection tube
14. Add 700 uL of DNA Wash Buffer
15. Centrifuge at maximum speed for 30 seconds
16. Discard the filtrate and reuse the collection tube
17. Repeat the steps for a second DNA Wash Buffer
18. Centrifuge the empty HiBind DNA mini column at maximum speed for 2 minutes to dry the column
19. Elute: Transfer the HiBind DNA mini column into a nuclease-free 1.5 mL microcentrifuge tube and label
20. Add 100 uL Elution Buffer heated to 70 degrees Celsius
21. Let sit at room temperature for 2 minutes
22. Centrifuge at maximum speed for 1 minute
23. Store eluted DNA at -20 degrees Celsius

Results/Data

After totalling the cell counts, adjusting for the ratio of solution:iodine, and averaging them, I calculated an average of 50 cells per μL. This means that of a 200 μL sample, there would be approximately 10,000 cells. The following 3 pictures are of the samples that I managed to take of each member of our group’s slides.

Conclusion

I spoke with Dr. Adair about my cell counts because they seemed to be a very high number of cells. While this is not exactly a great result, it is a better result than before, where it was hard to find ANY cells at all. I asked Dr. Adair about the number of cells and she said that atleast it shows that our protocol was successful in providing a substantial cell layer. I enjoyed this lab as it required a lot of diligence on our parts and paying careful attention to following the procedure outlined on the whiteboard and handout.