Week 5: DNA isolation from cheek cells
Cheek Cell DNA Template Preparation
Quick protocol
1. Label one 1.5 ml micro test tube with your
initials. Label one screwcap tube
containing 200 μl of InstaGene matrix with
your initials.
2. Obtain a cup containing saline solution
from your instructor. Pour the saline into
your mouth and rinse vigorously for 30
seconds. Expel the saline back into the cup.
3. Transfer 1 ml of your saline rinse into the
micro test tube (NOT the screwcap tube)
with your initials. If a P-1000 micropipet is
not available, carefully pour ~1 ml of your
saline rinse into your micro test tube (use
the graduations on the side of the micro test
tube to estimate 1 ml).
4. Spin your tube in a balanced centrifuge at
full speed for 2 minutes. When the
centrifuge has completely stopped, remove
your tube. You should see a match-head
sized pellet of whitish cells at the bottom
of the tube. If you don’t see a pellet of this
size, decant the saline, refill your tube with
more of your oral rinse, and repeat the
spin. Centrifuge
5. After pelleting your cells, pour off the
saline. Being careful not to lose your
pellet, blot your tube briefly on a paper
towel or tissue. It’s OK for a small amount
of saline (< 50 μl, about the same size as
your pellet) to remain in the bottom of the
tube.
6. Resuspend the pellet by vortexing or
flicking the tube so that no clumps of cells
remain.
7. Using a 2–20 μl adjustable-volume
micropipet set to 20 μl, transfer all of your
resuspended cells to the screwcap tube
containing InstaGene.
8. Screw the cap tightly on the tube. Shake or
vortex to mix the tube contents.
9. When all members of your team have
collected their samples, place the
tubes in the foam micro test-tube
holder, and incubate at
56°C for 10 minutes in a water
bath. At the 56°C, 10 min halfway point (5 minutes), shake or vortex the tubes
gently, then place back in the 56°C water bath for the remaining 5 minutes.
10. Remove the tubes, shake or vortex, and
place the tubes in a boiling water bath
(100°C). Incubate at 100°C for 5
minutes.
11. Remove the tubes from the boiling
water bath and shake or vortex the
contents to resuspend. Pellet the matrix
by spinning at 6,000 x g for 5 minutes (or
2,000 x g for 10 minutes) in a centrifuge.
Water bath
Centrifuge
100°C, 5 minWater bath
12. Store your screwcap tube in the
refrigerator until the next laboratory period
(or proceed to step 2 of Lesson 2).
Post-Lab Questions
Why is it necessary to chelate the metal ions from solution during the
boiling/lysis step at 100°C? What would happen if you did not use a chelating
agent such as the InstaGene matrix?
2. What is needed from the cells for PCR?
3. What structures must be broken to release the DNA from a cell?
4. Why do you think the DNA is stored cold with the InstaGene matrix after
boiling the samples?
