Nov 21, 2022
Salivary DNA Extraction
- Clemens Scherzer1,2,
- Bradley Hyman3,2,
- Charles Jennings1,2
- 1Brigham and Women's Hospital;
- 2Harvard Medical School;
- 3Massachusetts General Hospital
Protocol Citation: Clemens Scherzer, Bradley Hyman, Charles Jennings 2022. Salivary DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4k598vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47417
Keywords: DNA, extraction, salivary, saliva, ASAPCRN
Abstract
This protocol explains the Standard Operating Protocol for extracting salivary DNA.
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.
Materials
MATERIALS:
- OGR-600 Oragene DISCOVER saliva collection kit
- prepIT-L2P (catalog #: PT-L2P)
- DNA storage buffer: TE (10mM Tris-HCl, 1mL EDTA, pH 8.0) or similar solution
- 70% and 100% ethanol
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards. Gain all required consent and experimental approvals before beginning any procedures.
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)
Salivary DNA Extraction
Salivary DNA Extraction
4h 26m
4h 26m
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 air incubator for minimum 02:00:00 .
Note
Alternatively: Can incubate in 50 °C water bath Overnight the night before extraction or minimum 01:00:00 .
2h
Transfer the entire sample to a 15mL falcon tube by pouring.
Note volume of sample.
Volume of Sample:
Add 1/25th volume of PT-L2P and mix by vortexing for a few seconds.
Incubate On ice for 00:10:00 .
10m
Centrifuge 4500 rpm, Room temperature, 00:10:00 , max speed .
Carefully transfer most of the clear supernatant with a pipette to a fresh 50mL centrifuge tube.
Discard the pellet.
Add 1.2x volume of Room temperature 100% ethanol to the clear supernatant. Mix gently by inversion 10x.
Let the sample stand at Room temperature for 00:10:00 to allow DNA to fully precipitate.
10m
Centrifuge 4500 rpm, Room temperature, 00:10:00 , max speed .
Carefully pipette off the supernatant and discard it.
Avoid disturbing DNA pellet.
Add 1 mL 70% ethanol to the tube without disturbing the pellet.
Let stand at Room temperature for 00:01:00 , then gently swirl and completely remove the ethanol without disturbing the pellet.
1m
Let stand at Room temperature for 00:05:00 to allow remaining ethanol to evaporate.
5m
Rehydrate the DNA by adding 300 µL TE solution .
Mix by pipetting up and down 10x using wide-bore pipette tips.
Incubate at Room temperature for 01:00:00 or 4 °C Overnight .
2h
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 .
Note
Aliquots are labeled “DNA-SAL-01” and “DNA-SAL-02”.
DNA Sample Storage
DNA Sample Storage
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.