Week 8.1
5/19/09
Molecular Epidemiology Lab (1)
General overview of Lab (1):
DNA Extraction from Saliva Step 1 – Saliva Collection and Digestion
(1) Saliva collection
(2) Saliva sample digestion
DNA Extraction from Buccal cell Step 1 – Buccal Cell Collection and Digestion
(1) Buccal cells collection
(2) Centrifuge buccal cell samples
(3) Buccal cell samples digestion
Time Frame of Lab (1):
Time
Event
Note
10:00 ~ 10:10
Introduction
Introduction what we will do in the next 3 weeks;
wait for late persons; explain what we will do today
10:10 ~ 10:20
Saliva collection and
digestion
10:20 ~ 10:30
Buccal cell collection
10:30 ~ 10.55
Centrifuge samples (1)
How to use pipette and practice time.
10:55 ~ 11:20
Centrifuge samples (2)
Break time
11:20 ~ 11:40
Transfer sample to
Why buccal cells?
small tubes
11:40 ~ 11:50
Sample digestion
Week 8.1
5/19/09
 Instruction of DNA extraction from saliva
Step 1: Collection and Digestion
1. Spit saliva into the Oragene container until the amount of liquid saliva reaches the line that
indicated (around 2 ml).
2. Tighten the cap firmly and mix the saliva with the Oragene liquid (2 ml) via gently invert the
container several times.
3. Put parafilm around the cap and put it in the air incubator at 50oC for at least one hour and
then store it at room temperature until ready for further use.
 Instruction of DNA extraction from buccal cell
Step 1: Collection and Digestion
Buccal cell collection
1. Clean your mouth by Scope mouthwash.
2. Use toothbrush to gently brush each side of the cheeks 20 times.
3. Swish about 10 ml of Scope mouthwash for 30s and expel it to a 50ml conical tube.
4. Label your name on the conical tube.
Buccal cell sample centrifuging
1. Centrifuge the 50ml conical tube at 8000 rpm, 20C, for 25 minutes.
2. Discard the supernatant carefully. Make sure the buccal cell pellet is not discarded.
3. Transfer the buccal cell pellet into a 2ml microcentrifuge tube with a pipet. Clean the side of
the 50ml tube by 1ml TE buffer and transfer it into the 2ml microcentrifuge tube.
5. Centrifuge the 2ml microcentrifuge tube at 14000rpm for 15 minutes.
6. Discard the supernatant, then the sample is ready for digestion.
Buccal cell sample digestion
1. Add 200l digestion buffer and 10 l proteinase K into the 2ml microcentrifuge tube
contained buccal cell sample.
2. Resuspend the pellet with a pipet tip.
3. Close the tube tightly and put parafilm around the caps of the tube.
4. Vortex vigorously.
5. Incubate at 55 C overnight.