E.Z.N.A.® Tissue DNA Kit Protocol     2/15/2018

Materials:

• Tabletop microcentrifuge capable of 13,000 x g
• Nuclease-free 1.5 mL microcentrifuge tubes
• Heat block capable of 70°C
• Vortexer
• 100% ethanol
• 100% isopropanol
• PBS (or other wash buffer)

Mistakes:

We did not see a pellet after centrifuging the tube once, so we spun it again at 10,000g for 2 minutes.

Procedure:

Frozen cell samples should be thawed before starting this protocol.

1. Spin for 5 minutes at 3000g.
2. Remove the supernatant.
3. Add 1 mL 1x PBS, resuspend.
4. Spin for 5 minutes at 3000g.
5. Remove the supernatant.
6. Resuspend cells in 100 μL PBS.
7. Add 20 μL of iodine and 20 μL of cells into one tube.
8. Observe 5 2 μL drops and count the cells.
9. Add 25 μL OB Protease Solution. Vortex to mix.
10. Add 220 μL BL Buffer
11. Incubate at 70°C for 10 minutes in the heat block. Vortex the tube once during incubation.
12. Add 220 μL 100% ethanol. Vortex to mix thoroughly.
13. Insert a HiBind. DNA Mini Column into a 2 mL Collection Tube.
14. Transfer the entire sample to the HiBind. DNA Mini Column including any precipitates that may have formed.
15. Centrifuge at maximum speed (≥10,000 x g) for 1 minute.
16. Discard the filtrate and reuse the collection tube.
17. Wash and Dry
18. Add 500 μL HBC Buffer to the column.
19. Centrifuge at maximum speed for 30 seconds
20. After the HBC Buffer wash, discard the filtrate and collection tube.
21. Insert the HiBind® DNA Mini Column into a new 2 mL Collection Tube.
22. Add 700 μL DNA Wash Buffer.
23. Centrifuge at maximum speed for 30 seconds.
24. Discard the filtrate and reuse the collection tube.
25. Repeat the steps for a second DNA Wash Buffer wash step.
26. Centrifuge the empty HiBind® DNA Mini Column at maximum speed for 2 minutes to dry the column.
27. Transfer the HiBind® DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube.
28. Add 100μL Elution Buffer heated to 70°C.
29. Let sit at room temperature for 2 minutes.
30. Centrifuge at maximum speed for 1 minute.
31. Store eluted DNA at -20°C.

Data:

Cells counts: 66, 44, 40, 5, 7

Centrifuging our pellet did not work the first time around.

Conclusion:

This portion of the experiment was performed to extract the DNA. The next step is the PCR protocol. PCR is used to find the RNA primer for reading DNA. This is significant because the primers will help us unravel the DNA so it can be read and analyzed.

Errors: Our group accidentally threw out our filtrate which contained the DNA for this experiment.

Unexpected findings: We had to use 500 μL instead of 1x PBS.

Storage: Our sample is labelled KCLMKL and it is on the rack with the other samples in the lab room.