Dahlquist:Preparing Cheek Cell Lysate

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Overview

This protocol describes the steps necessary to obtain human cheek cell lysate for downstream PCR applications.

Recipes

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.

10% Chelex

Makes 10 mL. Store at room temperature.

• Weigh out 1 g of Chelex 100 (100-200 mesh, sodium form)
• Add 50 mM Tris to the Chelex, to make 10 mL of solution
• Adjust the pH to 11 using concentrated NaOH

Protocols

Prepare Cheek Cell Lysate

1. 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.
2. Pour all of the saline solution into your mouth and vigorously swish for 10 seconds. Save the tube for reuse in the next step.
3. Expel the saline solution into a cup. Then, carefully pour the saline mouthwash from the cup back into the 15 mL tube
4. 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.
5. 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.
6. Using the P1000 micropipettor, add 500 μL of 10% Chelex to the cell pellet:
   1. Cut the tip off of a blue tip to make a wider hole.
   2. Swirl or vortex the tube with the Chelex solution to resuspend the Chelex beads.
   3. Before the Chelex has had a chance to settle, transfer 500 μL to the tube with the cell pellet.
7. Resuspend the cells in the Chelex by vortexing. Hold the tube up to the light to confirm that no visible clumps of cells remain.
8. 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.
9. Incubate the 1.5 mL tube in a 100°C heat block for 10 minutes.
10. Cool the tube on ice for at least 1 minute.
11. 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.
12. 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.