Salivary DNA Extraction

Mix sample in the DNA Genotek kit by inversion and gentle shaking.

Weigh sample in original tube and subtract 6.81g to estimate saliva volume provided – amount of saliva collected is directly proportional to the amount of DNA recovered.

Incubate the sample at 50°C for minimum 2h 0m 0s.

Transfer the entire sample to a 15mL falcon tube by pouring.

Note volume of sample.

Add 1/25th volume of PT-L2P and mix by vortexing for a few seconds.

Incubate 50On ice for 0h 10m 0s.

Centrifuge 4500rpm.

Carefully transfer most of the clear supernatant with a pipette to a fresh 50mL centrifuge tube.

Discard the pellet.

Add 1.2x volume of 50Room temperature 100% ethanol to the clear supernatant. Mix gently by inversion 10x.

Let the sample stand at 50Room temperature for 0h 10m 0s to allow DNA to fully precipitate.

Centrifuge 4500rpm.

Carefully pipette off the supernatant and discard it.

Avoid disturbing DNA pellet.

Add 1mL to the tube without disturbing the pellet.

Let stand at 50Room temperature for 0h 1m 0s, then gently swirl and completely remove the ethanol without disturbing the pellet.

Let stand at 50Room temperature for 0h 5m 0s to allow remaining ethanol to evaporate.

Rehydrate the DNA by adding 300µL.

Mix by pipetting up and down 10x using wide-bore pipette tips.

Incubate at 50Room temperature for 1h 0m 0s or 4°C 1h 0m 0s.

Mix by pipetting up and down 10x using wide-bore pipette tips, then transfer the rehydrated DNA to 2 fresh 1.5mL low-retention microcentrifuge tubes (150uL each) for storage at -80°C.

Scan DNA samples into the Freezerworks Inventory Program and position in corresponding freezer.

Separate and store each DNA aliquot (DNA-01 and DNA-02 or DNA-SAL-01 and DNA-SAL-02) in two different local -80°C freezers.