Isolation of Buccal Cell DNA
This procedure is used to isolate individual DNA to be used in future PCR probes. Completely non-invasive and straight forward, it is a simple method to isolate small amounts of DNA from buccal cells.
15 mL sterile polypropylene centrifuge test tube
two sterile 1.5ml Eppendorf tube
5mL pipettor + sterile tips
1000 uL micropipettor + sterile tips
200 uL micropipettor + sterile tips
clinical centrifuge, balance
1.5 mL test tube holder (> 10 holes)
boiling water bath in 1000 mL beaker
SUPPLIES PER STUDENT:
10 mL 0.9% saline aliquoted into the 15 mL centrifuge tube
package of beverage straws
0.5 mL 10 % Chelex resin beads in sterile dH2O
Saline centrifuge tube
1.5 mL Eppendorf for separating Chelex
1.5 mL Eppendorf for storage of DNA
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.
Pour the 10 mL of saline solution into your mouth and vigorously swish against your cheeks for 10 seconds.
With a beverage straw, deliver the saline wash solution back into the labeled 15 mL polypropylene test tube.
SPIN DOWN CELLS
Place your test tube, with others, in a balanced array in the clinical centrifuge. Centrifuge at 2000 x g for 10 minutes. (Top speed, setting number 7 on the tabletop clinical centrifuge.)
The cells form a firm pellet below the saline supernatant.
SAVE THE PELLET, 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.
Drain all of the saline supernatant.
ADD CHELEX BEADS: Chelex is an ion exchange resin which removes polyvalent metal ions which might break down DNA during boiling or inhibit PCR reactions (next experiment).
Enlarge the aperture of the end of a 1000 uL pipetor tip (blue) by clipping off 2 mm from so that particulate matter will not stop it up. Use this prepared tip to resuspend the 10% suspension of Chelex resin beads by pipetting the beads in and out of the micropipettor. Before resin settles, pipet 500 uL of Chelex into the 15 mL tube containing your buccal cell pellet. Save pipet tip.
RESUSPEND CHEEK CELLS WITH CHELEX Using the same prepared blue tip, resuspend the cells in the pellet by pipetting in and out several times. (If the tip stops up, snip off 2 mm of the tip.)
Examine the suspension carefully to ensure that no visible clumps remain.
Using the same prepared tip, transfer a 500 uL aliquot of the cells and resin suspension to a clean 1.5 mL Eppendorf tube labeled with your name.
BOIL FOR 10 MINUTES: The cells are lysed and proteins denatured by exposing to 100 C for ten minutes: place your sample, along with other samples from the group, into a 1.5 mL floating test tube holder and float in a boiling water bath for 10 min.
Time the ten minutes in the boiling water bath.
CHILL ON ICE: After the heat treatment, 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 at top speed.
The chelex precipitates along with the denatured protein. The DNA is in the supernatant.
SAVE 200 uL CLEAR SUPERNATANT: Use a fresh pipet tip to transfer 200 uL of the clear supernatant to a clean 1.5 mL Eppendorf tube labeled with your:
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