Dahlquist:Preparing Cheek Cell Lysate
This protocol describes the steps necessary to obtain human cheek cell lysate for downstream PCR applications.
0.9% NaCl (Saline Solution)
NOTE: This will be used as a mouthwash, so food-grade salt (purchased at the grocery store), purified drinking water, and new, out-of-the-package, sterile 15 mL conical tubes will be used.
Makes 237 mL (8 ounces). Store at room temperature.
- Weigh out 2.13 g of NaCl (food-grade table salt) into a small food-grade cup on the balance.
- Carefully pour the salt into a newly-opened 8-ounce (237 mL) bottle of purified drinking water.
- Close lid and shake until salt is dissolved.
- Aliquot 10 mL each into new, out-of-the-package, sterile, 15 mL conical tubes.
Makes 10 mL. Store at room temperature.
- Weigh out 1 g of Chelex 100 (100-200 mesh, sodium form)
- Add 50 mM Tris to dry the Chelex, to make 10 mL of solution
- Adjust the pH to 11 using concentrated NaOH
Prepare Cheek Cell Lysate
- Use a sharpie to label the assigned ID number on a 15 mL conical tube containing 10 mL of a 0.9% sodium chloride (saline) solution.
- Pour all of the saline solution into your mouth and vigorously swish for 10 seconds. Save the tube for reuse in the next step.
- Expel the saline solution into a cup. Then, carefully pour the saline mouthwash from the cup back into the 15 mL tube
- Securely close the cap of the tube, and place the mouthwash tube in a balanced configuration with other tubes in the rotor of a clinical centrifuge. Spin at 3400 rpm for 10 minutes to pellet the cells on the bottom of the culture tube.
- Being careful not to disturb the cell pellet, pour off as much supernatant as possible into the cup. Place the tube with the mouthwash cell pellet on ice.
- Using the P1000 micropipettor, add 500 μL of 10% Chelex to the cell pellet:
- Cut the tip off of a blue tip to make a wider hole.
- Swirl or vortex the tube with the Chelex solution to resuspend the Chelex beads.
- Before the Chelex has had a chance to settle, transfer 500 μL to the tube with the cell pellet.
- Resuspend the cells in the Chelex by vortexing. Hold the tube up to the light to confirm that no visible clumps of cells remain.
- Transfer 500 μL of the resuspended mouthwash sample (including some of the Chelex beads) into a clean 1.5 mL microcentrifuge tube labeled with the assigned ID number.
- Incubate the 1.5 mL tube in a 100°C heat block for 10 minutes.
- Cool the tube on ice for at least 1 minute.
- Place the sample tube in a balanced configuration in a microcentrifuge rotor, and spin for 30 seconds at 10,000 rpm to pellet the Chelex beads at the bottom of the tube.
- Transfer 200 μL of the supernatant to a fresh 1.5 mL tube labeled with the ID number and place the tube on ice. Avoid transferring any of the Chelex pellet.
- This cheek cell lysate can be used immediately in a downstream PCR reaction, such as this one (use 2.5 μL lysate for a 25 μL reaction; 5 μL in a 50 μL reaction).
- Store lysate at -20°C.