DNA isolation protocol
– Chelex or InstaGene (Bio-Rad) matrix
1. Write your name on one clean 1.5 ml microcentrifuge tube and on a cup containing 10 ml saline (0.9% NaCI) solution.
2. Pour the saline solution into your mouth and vigorously rinse your mouth for 10 seconds.
3. Expel saline solution into the cup. Transfer 1 ml of your saline into the 1.5 ml microcentrifuge tube.
4. Place your microcentrifuge tube tube, together with other student samples, in a balanced pattern in the centrifuge and spin for 2 minutes at 12,000 x g.
5. Save the pellet of cheek cells! Carefully pour off supernatant into a beaker. Be careful not to disturb the cell pellet. It’s ok for a very small amount of saline (about the size of your pellet) to remain in the tube with your pellet.
6. Add 200 ul of Chelex solution or InstaGene matrix to your test tube: draw this suspension in and out of the pipet tip several times to suspend the resin beads. Before resin settles , rapidly transfer 200 µl of the suspension to the test tube. Note: you may have to cut the tip off of a 1 ml pipette tip for this step
7. Re-suspend cells by pipetting in and out several times – dispel all clumps of cells.
8. Place your sample in a floating tube rack in the 56°C water bath for 10 minutes
– at the halfway time (5 min) remove your tube and vortex gently
– place back in the water bath for the remaining 5 min.
9. Shake or vortex your sample, then place your microcentrifuge tube in a floating tube rack in the
100°C
water bath for 10 minutes. Cool on ice for 1 min.
10. Shake your sample briefly to resuspend any clumps of material, then centrifiuge for 5 min at 6,000 x g.
11.
Use a fresh tip to transfer 200 µl of the clear supernatant to a clean 1.5 ml tube labeled with your name and the date.
12. Store your sample on ice while you get ready for PCR