Pre-Lab Preparation

For each student, prepare and aliquot 10 ml 0.9% saline solution into a 15 ml polypropylene culture tube. The formula is 0.9 g NaCl per 100 ml distilled or deionized water.
For each student, aliquot 100 µl 10% Chelex® suspension into a 1.5 ml polypropylene tube. If using a thermal cyclcer for boiling, aliquot into the appropriate 0.5 ml or 0.2 ml PCR tube. Be sure to shake the stock tube (or draw liquid in and out of pipet tip several times) to re-suspend the Chelex beads each time before pipetting a student aliquot.
A boiling water bath can be made using a beaker and a hot plate. Affix a double layer of aluminum foil to the beaker, and use pencil or other point to make holes for 1.5 ml test tubes.

Procedure

1. Pour 10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish for 1 minute.
2. Expel saline solution into a paper cup.
   
3. Swirl to mix cells in the cup and transfer 1 ml (1000 µl) of the liquid to 1.5 ml tube.
   
4. Place your sample tube, together with other student samples, in a balanced configuration in a microcentrifuge, and spin for 1.5 minute.
   
5. Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.
   
6. Resuspend cells in remaining saline by pipetting in and out. (If needed, 30 µl of saline solution may be added to faciliate ressupension.)
   
7. Withdraw 30 µl of cell suspension, and add to tube containing 100 µl of Chelex. Vortex to mix.
   
8. Boil cell sample for 10 minutes. Use boiling water bath, heat block, or program thermal cycler for 10 minutes at 99°C. Then, cool tube briefly on ice (optional).
   
9. After boiling, vortex tube. Place in a balanced configuration in a microcentrifuge, and spin for 30 sec.
10. Transfer 30 µl of supernatant (containing the DNA) to clean 1.5 ml tube. Avoid cell debris and Chelex beads. This sample will be used for setting up one or more PCR reactions.
    
11. Store your sample on ice or in the freezer until ready to begin Part II.