Oct 26, 2021

Public workspaceDNA extraction from mouthwash samples V.2

This protocol is a draft, published without a DOI.
  • Ahmed A Shibl1,
  • Anique Ahmad1,
  • Tsedenia Denekew1,
  • Mamon Abd AlBaqi1,
  • Aashish Jha1
  • 1New York University, Abu Dhabi
Ahmed A Shibl
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Protocol Citation: Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Mamon Abd AlBaqi, Aashish Jha 2021. DNA extraction from mouthwash samples . protocols.io https://protocols.io/view/dna-extraction-from-mouthwash-samples-bzhip34eVersion created by Ahmed A Shibl
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: In development
We are still developing and optimizing this protocol
Created: October 26, 2021
Last Modified: October 26, 2021
Protocol Integer ID: 54538
Abstract
DNA extraction
Materials
QIAGEN - DNeasy PowerSoil Pro Kit
Ice
Vortex
BeadBeater
Centrifuge
Liquid N2
Before start
Set centrifuge to 4ºC
Keep CD2 solution on ice
Prepare and label collection tubes, microcentrifuge tubes, and MB spin columns
Thaw mouthwash samples on ice for Duration00:30:00

30m
Transfer desired volume into 1.5 mL or 2 mL eppendorfs
Amount1-2 mL

Centrifuge transferred samples at maximum speed at 4 ºC 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 high yield of DNA is required
Add Amount800 µL C CD1 and vortex to resuspend pellet

Spin down briefly
Transfer entire eppendorf content to PowerBead Pro tubes
Secure PowerBead Pro tubes onto the bead beater and run at maximum speed for Duration00:05:00

5m
Centrifuge the PowerBead Pro tubes at maximum speed for Duration00:01:30

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

10s
Centrifuge the 2 mL microcentrifuge tube at maximum speed for Duration00:01:30
1m 30s
Transfer the supernatant carefully without disturbing the pellet, into a clean 2 mL microcentrifuge tube
Add Amount600 µL 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 for Duration00:01:30
1m 30s
Discard flow-through and repeat step 13 to consume all the lysate
Place spin column onto new collection tube, add Amount500 µL EA, and centrifuge at maximum speed for Duration00:01:30
1m 30s
Discard flow-through and place spin column back into the collection tube
Add Amount500 µL C5 onto spin columns and centrifuge at maximum speed for Duration00:01:30
1m 30s
Discard flow-through and place spin column back into the collection tube
Centrifuge at maximum speed for Duration00:02:00 and place spin column into 1.5 mL eppendorf (elution) tube
2m
Add Amount50-100 µL nuclease-free water to the center of the spin column and leave at room temperature for around Duration00:05:00
5m
Centrifuge at maximum speed for Duration00:01:00 , quantify using Qubit, flash freeze, and store at -20/80 ºC
1m