TITLE: Salivary DNA Extraction
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for extracting salivary DNA.

[BEFORE_START]
DNA Q/C GOALS 
  1. Cary Concentration Assay 
        a. 260/280 = 1.8-2.0 
        b. Manual Puragene Extraction: 260 µg /mL (65 µg total) of DNA/subject 
        c. Automated QIAcube Extraction: 125 µg/mL (50 µg total) of DNA/subject 
  2. .7% Agarose Gel Electrophoresis 
        a. Human DNA = 23.13 kb with λ DNA-HindIII digest (NEB)

[GUIDELINES]
FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: Salivary DNA Extraction
1. Mix sample in the DNA Genotek kit by inversion and gentle shaking.
SECTION: Salivary DNA Extraction
1.1. 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.
SECTION: Salivary DNA Extraction
2. Incubate the sample at 50 °C for minimum 120 min.
 

SECTION: Salivary DNA Extraction
3. Transfer the entire sample to a 15mL falcon tube by pouring. 
SECTION: Salivary DNA Extraction
3.1. Note volume of sample. 
 

SECTION: Salivary DNA Extraction
4. Add 1/25th volume of PT-L2P and mix by vortexing for a few seconds.
SECTION: Salivary DNA Extraction
5. Incubate on ice for 10 min.

SECTION: Salivary DNA Extraction
6. Centrifuge 4500 rpm, 10 min.

SECTION: Salivary DNA Extraction
7. Carefully transfer most of the clear supernatant with a pipette to a fresh 50mL centrifuge tube.
SECTION: Salivary DNA Extraction
7.1. Discard the pellet.
SECTION: Salivary DNA Extraction
8. Add 1.2x volume of 50 Room temperature 100% ethanol to the clear supernatant. Mix gently by inversion 10x.

SECTION: Salivary DNA Extraction
9. Let the sample stand at 50 Room temperature for 10 min to allow DNA to fully precipitate.

SECTION: Salivary DNA Extraction
10. Centrifuge 4500 rpm, 10 min.

SECTION: Salivary DNA Extraction
11. Carefully pipette off the supernatant and discard it.
SECTION: Salivary DNA Extraction
11.1. Avoid disturbing DNA pellet.
SECTION: Salivary DNA Extraction
12. Add 1 mL to the tube without disturbing the pellet.

SECTION: Salivary DNA Extraction
12.1. Let stand at 50 Room temperature for 1 min, then gently swirl and completely remove the ethanol without disturbing the pellet.

SECTION: Salivary DNA Extraction
13. Rehydrate the DNA by adding 300 µL.

SECTION: Salivary DNA Extraction
12.2. Let stand at 50 Room temperature for 5 min to allow remaining ethanol to evaporate.

SECTION: Salivary DNA Extraction
14. Incubate at 50 Room temperature for 60 min or 4 °C 60 min.
SECTION: Salivary DNA Extraction
13.1. Mix by pipetting up and down 10x using wide-bore pipette tips.
SECTION: Salivary DNA Extraction
15. 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.
 


SECTION: DNA Sample Storage
16. Scan DNA samples into the Freezerworks Inventory Program and position in corresponding freezer.
SECTION: DNA Sample Storage
17. 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.