Oct 13, 2022

Public workspaceDNA extraction from mouthwash samples using Qiagen DNeasy PowersoilPro Kit

DOI
dx.doi.org/10.17504/protocols.io.eq2ly7dyrlx9/v1
  • Ahmed A Shibl1,
  • Anique Ahmad1,
  • Tsedenia Denekew1,
  • Mamon Abd AlBaqi1,
  • Aashish R Jha1
  • 1New York University, Abu Dhabi
  • Aashish R Jha: jhaar@nyu.edu
nyuad.geneticheritagegroup
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly7dyrlx9/v1
Protocol Citation: Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Mamon Abd AlBaqi, Aashish R Jha 2022. DNA extraction from mouthwash samples using Qiagen DNeasy PowersoilPro Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7dyrlx9/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: September 18, 2022
Last Modified: October 13, 2022
Protocol Integer ID: 70195
Abstract
This protocol describes how to extract DNA from mouthwash samples.
Materials
QIAGEN -DNeasy PowerSoil Pro Kit
Ice
Vortex
Omni Bead Ruptor Elite bead beater
Centrifuge
Liquid N2

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


Thaw the sample on ice for Duration00:30:00


30m
Transfer the desired sample volume Amount1-2 mL into a 1.5mL or 2mL eppendorf tube

Centrifuge the transferred sample at maximum speed (Centrifigation17000 x g ) at 4°C for Duration00:10:00

10m
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 Amount800 µL 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 (Centrifigation17000 x g ) for Duration00:05:00

5m
Centrifuge the PowerBead Pro tube at maximum speed (Centrifigation17000 x g ) for Duration00:01:30

1m 30s
Transfer the supernatant carefully, without disturbing the pellet, into a clean 2mL microcentrifuge tube
Add Amount200 µL of CD2 into the 2mL microcentrifuge tube and vortex for Duration00:00:10

10s
Centrifuge the 2mL microcentrifuge tube at maximum speed (Centrifigation17000 x g ) for Duration00:01:30

1m 30s
Transfer the supernatant carefully without disturbing the pellet, into a clean 2mL microcentrifuge tube
Add Amount600 µL of CD3 into the 2 mL microcentrifuge tube and vortex for Duration00:00:10

10s
Load Amount650 µL of the lysate onto MB spin columns and centrifuge at maximum speed (Centrifigation17000 x g ) for Duration00:01:30

1m 30s
Discard flow-through and repeat step 13 to consume all the lysate
Place the spin column onto a new collection tube, add Amount500 µL of EA, and centrifuge at maximum speed (Centrifigation17000 x g ) for Duration00:01:30

1m 30s
Discard flow-through and place the spin column back into the collection tube
Add Amount500 µL of C5 onto the spin column and centrifuge at maximum speed (Centrifigation17000 x g ) for Duration00:01:30

1m 30s
Discard flow-through and place the spin column back into the collection tube
Centrifuge at maximum speed (Centrifigation17000 x g ) for Duration00:02:00 and place spin column into 1.5mL eppendorf (elution) tube

2m
Add Amount50-100 µL of nuclease-free water to the center of the spin column and leave at room temperature for Duration00:05:00

5m
Centrifuge at maximum speed (Centrifigation17000 x g ) for Duration00:01:00 , quantify using Qubit, flash freeze, and store at -20/80 °C

1m