Molecular Epidemiology Lab (3)
Lab (2) Review:
DNA Extraction from Buccal Cell Step 2 -- Extraction
(1) Extract DNA Part I
(2) Leave in freezer
General overview for Lab (3):
DNA Extraction from Buccal Cell Step 3 -- Purification
(1) Extract DNA Part II – Purification
(2) Dissolve DNA with storage buffer
DNA Extraction from Saliva Step 2 – Extraction and Purification
(1) Extract and purify DNA with Oragene Purifier kit
(2) Dissolve DNA with storage buffer
Time Frame of Lab (3):
Time / Event / Note10:00 ~ 10:05 / Introduction / Introduction what we will do today
10:05 ~ 10:25 / 1st centrifuge
10:25 ~ 10:30 / 70% alcohol step
10:30 ~ 10:40 / 2ND centrifuge
10:40 ~ 10:45 / 100% alcohol step
10:45 ~ 11:00 / 3RD centrifuge / Remove additional ethanol and dissolve DNA in TE buffer, 5 minutes break
11:00 ~ 11:10 / Saliva Purification
step 1 / Transfer Saliva samples to small tubes, add Oragene Purification kit
11:10 ~ 11:20 / Incubate tubes on ice
11:20 ~ 11:35 / Saliva Purification step 2 / Centrifuge, transfer supernatant to new tubes, add 95% ethanol
11:35 ~ 11:45 / Wait for 10 minutes / at room temperature
11:45 ~11: 55 / Dissolve DNA / Centrifuge, discard the supernatant, remove additional ethanol and dissolve DNA in TE buffer
Instruction of DNA extraction from Buccal cell
Step 3: Extraction (2) – Purification
1. Spin the samples at high speed in a microcentrifuge for 20mins.
2. Carefully remove the supernatant without touching the pellet or the area where the pellet is expected to be.
3. Add 1ml 70% ethanol, invert several times, spin for 10 min at high speed and carefully remove the supernatant.
4. Add 1ml of 100% ethanol, invert several times, spin for 5-10 min and carefully remove the supernatant as completely as possible. Put it in the air incubator to further remove the ethanol if needed.
5. Resuspend the pellet in 200-400 l storage buffer and store the samples at 4oC.
Instruction of DNA extraction from Saliva
Step 2: Extraction and Purification
1. Transfer 1ml of the Oragene/saliva sample into a 1.5 ml microcentrifuge tube. The rest of the Oragene/saliva sample can be stored at room temperature until ready for further use.
2. Add 40 l of Oragene Purifier to the tube and mix gently by inversion. The sample will become turbid as impurities are precipitated.
3. Incubate the tubes on ice for 10 min.
4. Centrifuge for 3 minutes at high speed. Carefully pipet the clear supernatant into a fresh microcentrifuge tube without disturbing the pellet. Discard the pellet.
5. Add 1ml (equal volume) of room temperature 95% ethanol to the supernatant and mix gently by inversion. Invert at least 5 times. A colt of DNA may be visible.
6. Let the solution stand for 10 minutes at room temperature so that the DNA is fully precipitated. Do not incubate at -20oC because impurities will co-precipitate with the DNA.
7. Centrifuge for 3 min at high speed. Discard the supernatant without disturbing the DNA pellet.
8. Once all of the ethanol has been removed, dissolve the DNA pellet in 200 l storage buffer.