DNA extraction from mouthwash samples using Qiagen DNeasy PowersoilPro Kit
Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Mamon Abd AlBaqi, Aashish R Jha
Abstract
This protocol describes how to extract DNA from mouthwash samples.
Before start
Set centrifuge to 4ºC
Keep CD2 solution on ice or at 4ºC
Prepare and label collection tubes, microcentrifuge tubes, MB spin columns, and powerbead tubes
Steps
Thaw the sample on ice for 0h 30m 0s
Transfer the desired sample volume 1-2mL into a 1.5mL or 2mL eppendorf tube
Centrifuge the transferred sample at maximum speed (17000x g,0h 0m 0s ) at 4°C for 0h 10m 0s
Discard the supernatant carefully without disturbing the pellet
Repeat Step 1.1 if more volume is needed to see a pellet or a higher yield of DNA is required
Add 800µL of CD1 and vortex to resuspend pellet
Spin down briefly
Transfer entire eppendorf content in a PowerBead Pro Tube
Secure PowerBead Pro tube onto the bead beater and run at maximum speed (17000x g,0h 0m 0s ) for 0h 5m 0s
Centrifuge the PowerBead Pro tube at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 30s
Transfer the supernatant carefully, without disturbing the pellet, into a clean 2mL microcentrifuge tube
Add 200µL of CD2 into the 2mL microcentrifuge tube and vortex for 0h 0m 10s
Centrifuge the 2mL microcentrifuge tube at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 30s
Transfer the supernatant carefully without disturbing the pellet, into a clean 2mL microcentrifuge tube
Add 600µL of CD3 into the 2 mL microcentrifuge tube and vortex for 0h 0m 10s
Load 650µL of the lysate onto MB spin columns and centrifuge at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 30s
Discard flow-through and repeat step 13 to consume all the lysate
Place the spin column onto a new collection tube, add 500µL of EA, and centrifuge at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 30s
Discard flow-through and place the spin column back into the collection tube
Add 500µL of C5 onto the spin column and centrifuge at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 30s
Discard flow-through and place the spin column back into the collection tube
Centrifuge at maximum speed (17000x g,0h 0m 0s ) for 0h 2m 0s and place spin column into 1.5mL eppendorf (elution) tube
Add 50-100µL of nuclease-free water to the center of the spin column and leave at room temperature for 0h 5m 0s
Centrifuge at maximum speed (17000x g,0h 0m 0s ) for 0h 1m 0s , quantify using Qubit, flash freeze, and store at -20/80 °C
