Check cell DNA isolation via The E.Z.N.A.® Tissue DNA Kit

Goal: Isolation of genomic DNA for PCR analysis. You will dislodge cheek cells from your mouth by swishing a saline solution in your mouth. To separate your cheek cells from the saline solution, you will centrifuge the mixture, which yields a cell pellet at the bottom of the tube. After lysing open the cells, you’ll isolate the DNA contained in the cells. The key to this system is a column containing a HiBind matrix that specifically, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or a low salt buffer.

Reagents for cell collection:

• 0.9% saline solution, 5 mL
• Paper cup
• Chill PBS to 4°C.

1. Pour saline solution into your mouth, and vigorously rinse your cheek pockets for 30 seconds. You want to try to remove some cells from the inside of your cheeks, so you can scrape your teeth against your cheeks, but don’t bite too hard!
2. Expel saline solution into the paper cup.
3. Swirl the cup gently to mix cells that may have settled to the bottom. Use a micropipette with a fresh tip to transfer ~1400 μL of the solution into a labeled microfuge tube.
4. Place your tube in a balanced microcentrifuge, and spin for 4 min at 1,200 xg. During this time, label your tubes for the DNA isolation protocol below.
5. Your DNA is contained in the cells at the bottom of the tube. Carefully pipette off the supernatant, but be careful not to disturb the cell pellet at the bottom of the tube.  The supernatant you removed can be discarded in the sink.
6. Add 200 uL cold 1X PBS and resuspend the cell pellet in the PBS by pipetting in and out until the mixture is homogenous. Pipette slowly to minimize bubbles.

Preparation for DNA isolation kit:

• Set heat block to 70 °C
• Heat Elution Buffer to 70 °C (you need 100 uL per isolation)

1. Add 25 μL Proteinase K Solution to your cell solution. Vortex to mix thoroughly.
2. Add 220 μL BL Buffer. Note: A wispy precipitate may form upon the addition of BL Buffer. This does not interfere with DNA recovery.
3. Incubate at 70°C for 10 minutes. Briefly vortex the tube once during incubation.
4. Add 220 μL 100% ethanol. Vortex to mix thoroughly.
5. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
6. Transfer the entire sample from Step 4 to the HiBind® DNA Mini Column including any precipitates that may have formed.
7. Centrifuge at maximum speed (≥10,000g) for 1 minute.
8. Discard the filtrate and reuse the collection tube.
9. Add 500 μL HBC Buffer (that has been diluted with 100% isopropanol before use) to the column.
10. Centrifuge at maximum speed for 30 seconds.
11. Discard the filtrate and collection tube.
12. Insert the HiBind® DNA Mini Column into a new 2 mL Collection Tube.
13. Add 700 μL DNA Wash Buffer (previously diluted with 100% ethanol before use)
14. Centrifuge at maximum speed for 30 seconds.
15. Discard the filtrate and reuse the collection tube.
16. Repeat Steps 13-15 for a second DNA Wash Buffer wash step.
17. Centrifuge the empty HiBind® DNA Mini Column at maximum speed for 2 minutes to dry the column. Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.
18. Transfer the HiBind® DNA Mini Column into a NEW nuclease-free 1.5 mL microcentrifuge tube.
19. Add 50 μL Elution Buffer heated to 70°C to the center of the column.
20. Let sit at room temperature for 2 minutes.
21. Centrifuge at maximum speed for 1 minute to elute DNA.
22. Repeat Steps 19-21 for a second elution step with another 50 μL (elute into the same tube as the first elution).
23. Store eluted DNA at -20°C.