ISOLATION OF BUCCAL CELL DNA

9 January 2001, 13 Feb 01

modified from the protocol of Sonia Wallman

David B. Fankhauser, Professor of Biology and Chemistry

EQUIPMENT: SUPPLIES PER STUDENT:

15 mL sterile polypropylene test tube

two sterile 1.5ml Eppendorf tube

5mL pipettor + sterile tips

1000 無 micropipettor + sterile tips

200 無 micropipettor + sterile tips

clinical centrifuge, balance

1.5 mL test tube holder (> 10 holes)

boiling water bath in 1000 mL beaker

microcentrifuge

10 mL 0.9% saline

0.5 mL 10 % Chelex resin beads in sterile dH2O

HARVEST BUCCAL CELLS: Label a 15 mL polypropylene test tube and the top of a 1.5ml Eppendorf tube with your name and/or seat number.
Pipet 10 mL of 0.9% saline into the test tube, using 5 mL pipettor.
Pour the 10 mL of saline solution into your mouth and vigorously swish for 10 seconds.
Expel saline solution back into the labeled 15 mL polypropylene test tube over the sink.
Place your test tube, with others, in a balanced array in the clinical centrifuge.
SPIN DOWN CELLS Centrifuge at 2000 x g for 10 minutes. (Top speed, setting number 7 on the tabletop clinical centrifuge.)
DISCARD THE SUPERNATANT by decanting into the sink with running water, taking care not to disturb pelleted cheek cells at the bottom of the tube.
Set micropipettor to 500 無.
ADD CHELEX BEADS: Use micropipettor to resuspend Chelex resin beads, by pipetting the beads in and out of the micropipettor. Before resin settles, pipet 500 無 of Chelex to the 15 mL tube containing your cheek cell pellet. Save pipet tip.
RESUSPEND CHEEK CELLS WITH CHELEX by pipetting in and out several times. Examine to confirm that no visible clumps remain.
Pipet in and out several times to resuspend cells and resin, then transfer 500 無 of the suspension of cells and resin to a 1.5 mL Eppendorf tube labeled with your name. (If the tip stops up, snip off 2 mm of the tip to enlarge the aperture.)
BOIL FOR 10 MINUTES: Place your sample, along with others, into a 1.5 mL test tube holder and place in a boiling water bath for 10 min.
CHILL ON ICE: Transfer all samples to crushed ice.
SPIN DOWN CHELEX: Place your chilled sample, along with others, in a balanced array in a microcentrifuge and spin for 30 to 60 seconds.
SAVE 200 無 CLEAR SUPERNATANT: Use a fresh pipet tip to transfer 200 無 of the clear supernatant to a clean 1.5 mL Eppendorf tube labeled with your name, date, and "cheek DNA." Take care not to pick up any of the cheek cell debris or resin from the bottom of the tube.
Store your sample for a few minutes or hours on crushed ice or for days at -20 degrees Centigrade until you are ready to proceed to Set up and run PCR reaction.