I: DNA Isolation by Saline Mouthwash
solution (0.9% NaCl),
10% Chelex«, 100 Ál
ml test tube, polypropylene
1.5 ml test tubes, polypropylene
1 ml transfer pipet or
100-1,000 Ál micropipet and tip
Cup of ice (optional)
Boiling water bath, heat block, or Thermal cycler
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 Ál 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.
10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish
for 1 minute.
saline solution into a paper cup.
to mix cells in the cup and transfer 1 ml (1000 Ál) of the liquid to
1.5 ml tube.
your sample tube, together with other student samples, in a balanced
configuration in a microcentrifuge, and spin for 1.5 minute.
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.
cells in remaining saline by pipetting in and out. (If needed, 30 Ál
of saline solution may be added to faciliate ressupension.)
30 Ál of cell suspension, and add to tube containing 100 Ál of Chelex.
Vortex to mix.
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
boiling, vortex tube. Place in a balanced configuration in a microcentrifuge,
and spin for 30 sec.
30 Ál 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.
your sample on ice or in the freezer until ready to begin Part II.