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Cancer Research Center of Hawaii
MOUTHWASH PROTOCOL FOR BUCCAL CELL COLLECTION
Lum, A., Le Marchand, L. 1998. A simple mouthwash method for obtaining genomic DNA in molecular epidemiologic studies. Cancer Epidemiol. Biomarkers Prev. 7: 719-24.
For PDF version of this protocol click here: buccal_protocol.pdf

PURPOSE
Research study participants who refuse the blood donation are requested to provide a buccal cell specimen. Subjects may collect the sample themselves and mail it back to the laboratory. The cells are collected with a simple "swish-and-spit" technique using commercial mouthwash.

SUPPLY FOR COLLECTION KITS:
COLLECTION AND STORAGE
A collection kit is given or mailed to the subject along with an instruction sheet (see attached). The description of the procedure is self-explanatory.
The sample should be frozen at -20° C upon reception.
Samples should be stable for at least six-months (Lum et al., CEBP 1998;7:719-24).

ISOLATION OF DNA (LONG PROTOCOL)
Reference:
Walsh, D.J., et al. Isolation of DNA from Saliva and Forensic Science Samples Containing Saliva, Journal of Forensic Sciences, JFSCA, Vol. 37, No. 2, March 1992, pp. 387-395.
(DNA isolation modified from above mentioned reference)
1. Place mouthwash sample in 50 ml conical tube (Falcon tube). Centrifuge at 2700 rpm for 15 min. Dump supernatant and resuspend pellet in 25 ml T10E10 to remove residual mouthwash from sample.
T10E10:
10 ml 1M Tris/HCl pH 8.0
20 ml 0.5M EDTA pH 8.0
Total vol = 1 liter
Centrifuge sample at 2700 rpm for 15 min. Dump super.
2. Resuspend pellet in 700 ml lysis buffer.
Lysis buffer:
10 mM Tris/HCl pH 8.0
10 mM EDTA pH 8.0
0.1 M NaCl
2% SDS
Transfer lysis buffer containing the pellet into a 2.0 ml microcentrifuge tube.

3. Add 35 ml 20 mg/ml Proteinase K to each sample. Vortex to mix sample and incubate at 58° C for 2 hr.
4. Add 700 ml 1:1 phenol:chloroform. Vortex for 10 sec, and centrifuge at 14,000 rpm for 2 min. Remove super and place in new 2 ml tube.
5. Add 700 ml chloroform to each sample. Vortex for 10 sec and centrifuge at 14,000 rpm for 2 min. Remove super and place in new 2 ml tube.
6. Precipitate DNA with 1/10 volume of 3M NaOAc pH 6.0. Invert to mix. Add 2 volumes of 100% EtOH. Invert to mix and leave at -20° C for 2 hr/overnight.
7. Spin at 10,000 rpm for 10 min. Dump super.
8. Wash pellet with 500 ml 70% EtOH. Spine at 14,000 rpm for 2 min. Dump super. Dry pellet using SpeedVac for 1 min. Resuspend in TE. The size of the pellet will determine the amount of TE used to resuspend. Calculate the concentration of your genomic DNA using the GeneQuant.

DNA EXTRACTION USING QIAGEN TISSUE KIT (Cat. #: 29304)
1. Spin 10 ml Scope sample at 2300 rpm for 15 min. Dump supernatant.
2. Rinse pellet with 25 ml T10E10 (10 ml 1M Tris/HCl pH 8.0). Vortex to resuspend pellet. Spin. Dump super. Remove residual TE with pipette.
3. Add 360 ul Buffer ATL to pellet. Add sample to 2 ml microcentrifuge tube. Add 20 ul Proteinase K (included in kit). Vortex. Incubate at 55 ° C for 2 hours. Add an additional 20 ul Proteinase K and incubate at 55 ° C 1hr/overnight. Overnight is for optimal DNA recovery.
4. Add 400 ul of Buffer AL. Vortex. Incubate at 70 ° C for 10 min.
5. Add 420 ul 100% EtOH. Vortex. Put spin column in 2 ml capture tube.
6. Add sample into spin column. Spin at full speed 1 min. Dump filtrate. Repeat with rest of sample.
7. Place spin column in clean capture tube. Add 500 ul Buffer AW. Spin at full speed for 1 min. Repeat.
8. Remove column to clean 1.5 ml tube. Add 200 ul Buffer AE (preheated to 70 ° C) to column. Incubate assembly to 70 ° C for 5 min. Spin at full speed for 1 min.
9. Re-add filtrate to column. Incubate again at 70 ° C for 5 min. Spin and calculate concentration.

DIRECTIONS FOR MOUTHWASH
The purpose of this simple procedure is to collect some loose cells in your mouth. It involves the use of Scope (a commercial brand of mouthwash) that is provided to you in a small sealed bottle. You are to swish one tablespoon of Scope around in your mouth for one minute, then spit it back into the container (also enclosed). Please follow the directions below.
1. Brush your teeth as you usually do, and wait about 30 minutes. Preferably, do not eat or drink anything other than water during this time.
2. Be sure to write the date on the container’s label.
3. Open the bottle of Scope by removing the printed safety band, and by pushing down while turning the cap. Remove the empty container from the plastic bag. Pour some mouthwash into the container up to the black mark on the side of the container.
4. About a half an hour after you brush your teeth, pour the mouthwash from the container into your mouth, without swallowing.
5. Swish the mouthwash around in your mouth vigorously for 60 seconds. Watch the clock while you do this. It is important that you do not shorten the time you swish the mouthwash, but there is no harm in doing it longer than 60 seconds.
6. Spit the mouthwash back into the container. Replace the cover on the container and screw it on tightly. Please keep the remaining portion of Scope for your own use.
7. Place the container with the sample back into the plastic bag. Push the air out of the bag before sealing it. Place the bag into the small bubbled envelope. This envelope has our address on it and carries the required postage. Please drop the return envelope into the mail within 24 hours after collection.

THANK YOU


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