11 DNA Isolation from saliva protocol

DNA Isolation from saliva protocol

Purpose: Extract DNA from your cheek cells

Overview of method:

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. Once you have these cheek cells collected, you will transfer them into a solution containing 10% Chelex resin. This mixture is placed into boiling water to lyse the cells and release the DNA. The Chelex beads bind to magnesium ions (Mg²⁺). These ions often serve as co-factors for nuclease enzymes that can degrade DNA. By removing Mg²⁺ with the Chelex resin, DNA degradation is prevented.

The mixture containing DNA, cell debris, and Chelex is centrifuged to pellet the cell debris and Chelex. The DNA will remain in the supernatant (the top layer), which allows you to separate DNA from the Chelex and cell debris. Chelex DNA isolation is quick and easy; however, the DNA will NOT be pure. The resulting DNA contains proteins and even nucleic acids from bacteria in your mouth. Generally, these contaminants do not inhibit PCR, since PCR primers are designed to amplify specific genomic sequences.

Common issues:

• Chelex resin is a potent inhibitor of PCR, since DNA Polymerase also uses Mg²⁺ as a cofactor. Therefore, it is critical that you do not transfer ANY Chelex resin into your final DNA sample or your PCR reaction will fail.
• Chelex resin sediments VERY quickly. Therefore, it is critical that you vortex Chelex stock solutions before EACH time you pipette the solution.

Reagents:

• 0.9% saline solution, 5 mL
• 10% Chelex stock (VORTEX BEFORE TAKING EVERY ALIQUOT)
• 5 sterile microfuge tubes per person
• Paper cup

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 1000 μL of the solution into a labeled microfuge tube.
4. Place your tube in a balanced microcentrifuge, and spin for 90 seconds at full speed.
5. Your DNA is contained in the cells at the bottom of the tube. Carefully pipette off the supernatant until you reach the 0.1 (100 uL) mark on the tube (You want to have 100 uL of liquid left in your tube). 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. Use a P200 pipette to gently resuspend the cell pellet in the remaining saline by pipetting in and out until the mixture is homogenous. Pipette slowly to minimize bubbles.
7. Withdraw 30 μL of cell suspension, and add it to a new microfuge tube. Repeat this step and add 30 uL of cell suspension to a second microfuge tube. (You are preparing two duplicate samples so you have plenty of DNA to use this semester)
8. VORTEX the 10% Chelex stock until it is fully homogenous. This will take ~1 min of vortexing. THEN, quickly use a P1000 to transfer 100 μL of the 10% Chelex suspension to each tube containing 30 uL of cell suspension. Using the wider tip of the P1000 is necessary to avoid clogging the pipette tip with resin.
9. VORTEX the 10% Chelex stock before transferring 100 uL of the Chelex suspension to your second tube of cells.
10. Place lid-locks on your two tubes. Use a heat block to boil your samples at 99 °C for 10 minutes
11. After boiling, let tubes cool for 1 minute, and then vigorously vortex for 5 seconds. Let tubes sit upright for 1 minute to sediment Chelex.
12. Place your tubes in a balanced microcentrifuge, and spin for 90 seconds at full speed.
13. Your DNA is now located in the supernatant. The pellet contains the Chelex resin and PCR contaminants you need to remove. Use a micropipette with a fresh tip to transfer ~30 μL of the clear supernatant into a clean microfuge tube. Be careful to avoid pipetting any cell debris and Chelex resin.
14. Repeat with your second tube, and transfer 30 μL of your supernatant into a second new tube.
15. Label the cap and side of the tube. This DNA sample will be used for setting up one or more PCR reactions.
16. Store your samples at –20 °C until you are ready to continue.