Apr 25, 2022
DNA Extraction from Modern Dental Plaque on Gauze
Forked from DNA Extraction from Modern Dental Calculus
- 1Friedrich-Schiller Universität Jena;
- 2Max Planck Institute for Evolutionary Anthropology
Protocol Citation: Franziska Aron, Irina Velsko 2022. DNA Extraction from Modern Dental Plaque on Gauze. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lypejrlx9/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: March 08, 2021
Last Modified: April 25, 2022
Protocol Integer ID: 47971
Keywords: DNA, extraction, dental plaque
Abstract
Protocol for DNA extraction from modern dental plaque dried on gauze samples for Illumina sequencing. This protocol uses the Qiagen PowerSoil DNA extraction kit, but applies modifications to improve DNA recovery. Specifically, solution C2 is not used to avoid uneccessary DNA loss during inhibitor removal.
Guidelines
Definitions
Stock-aliquot refers to a personal 'stock' (e.g. in a 50ml Falcon Tube) of reagents you can use across multiple sessions of this protocol. An 'aliquot' refers to a sub-aliquot of the stock, that is used for a single session of this specific protocol.
Protocol Specific Guidelines
This protocol requires the use of a Biological Safety Laboratory level S2 due to handling of human substrates.
Materials
Consumables
1.5ml Eppendorf DNA LoBind tubes
Qubit™ dsDNA BR Assay KitThermo FisherCatalog #Q32853
EDTA (0.5 M) pH 8.0Life TechnologiesCatalog #AM9261
Water HPLC Plus Merck Millipore SigmaCatalog #34877-2.5L-M
2 ml LoBind TubesEppendorfCatalog #0030108078
DNeasy PowerSoil Kit (100)QiagenCatalog #12888-100
Rotilabo®-MikropistilleCarl RothCatalog #YE15.1
Proteinase K from Tritirachium albumMerck Millipore SigmaCatalog #P2308-10MG
Roti®-Tape-MarkierbandCarl RothCatalog #AK65.1
Falcon 50 mlgreiner bio-oneCatalog # 210261
Equipment
Centrifuge 5424 R refrigerated with Rotor FA-45-24-11 rotary knobs 120 V/50 – 60 Hz (US)Eppendorf CentrifugeCatalog #5404000537
Tube rotatorVWR international LtdCatalog #444-0500P Tube rotatorVWR international LtdCatalog #444-0500P
Vortex-Genie® 2 EU-SteckerVwrCatalog #444-5900P
Equipment
Bead Ruptor Elite
NAME
Omni International
BRAND
19-040E
SKU
Generic Reagents
Paper towels or tissues
Safety warnings
Location
Work must be performed in BSL-S2 safety lab (Germany, or equivalent for your country).
Wear nitrile gloves, a lab coat and lab safety glasses.
Reagents
Proteinase K
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.
- H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.
- H335 May cause respiratory irritation.
Kits
Check manufacturer's safety information for the High Pure Viral Nucleic Acid Large Volume Kit used in this protocol.
Before start
Planning
This protocol requires the use of a Biological Safety Laboratory level S2 due to handling of human substrates.
This protocol takes ~1 day.
Prepare a cool tube-rack for 1.5ml and 2ml tubes by placing it at +4°C for the DNA-clean up. This will speed up reaction time with a 'cold start'.
Modern dental plaque samples should be stored at least at +4°C or preferably at -20°C to preserved DNA after sampling.
Check waste disposal guidance for all reagents in this protocol against your corresponding laboratory regulations.
Equipment
Make sure all necessary equipment is available (see Materials).
Abbreviations
HPLC = High Performance Liquid Chromatography (i.e., HPLC-grade water)
Controls
Consider taking along a positive control (sample of known performance) and a negative control (tube with HLPC-grade water instead of DNA) in order to assess the performance of the protocol and the level of background contamination. Take into account these two extra samples in your calculations for buffer preparations.
Preparation Day 1
Preparation Day 1
Place a 1.5ml/2ml tube rack at 4 °C for the incubation steps on the final days
Label for each sample one 2ml-Eppendorf Safe-Lock DNA LoBind-Tube
and one 1.5ml-Eppendorf Safe-Lock DNA LoBind-Tube
Place solution C6 in an incubator to pre-heat it to 37 °C .
Preparation for Final Day
Preparation for Final Day
Retrieve the spin filter columns (provided in the kit) from fridge to warm them up to Room temperature for use at step 25
Note
"provided in the kit" refers to: DNeasy PowerSoil Kit (100) Cat No./ID: 12888-100
A spin filter column is a spin filter unit sitting in a collection column. The spin filter unit contains the membrane.
Label for each sample one Power Bead tube, one spin filter column and three 2ml collection tubes (all provided in the kit).
Note
Be sure to label both the top and sides of the PowerSoil bead tube. The labeling on the top may be rubbed off during bead-beating with the Omni BeadRuptor.
Sample Preparation
Sample Preparation
Sterilize scissors by wiping with 75% ethanol, and then wiping them with HPLC water, then wipe dry. Use the scissors to clip a small piece of gauze with plaque.
Note
Do not take more than half of the plaque, in case more is needed for another extraction later.
Place the piece of gauze with plaque in a PowerSoil bead tube using forceps sterilized by wiping with 75% ethanol, and then wiping them with sterile water.
Safety information
Change gloves and sterilize scissors and forceps between each sample.
Make two extraction blank PowerSoil bead tubes per extraction batch, one with nothing added, and a second with a small section of sterile gauze added.
DNA_Clean_up
DNA_Clean_up
Invert the PowerSoil bead tubes to mix and fully submerge gauze with plaque.
Add 60 µL C1 solution, invert several times to mix.
Add 25 µL proteinase K (10 mg/mL ), and rotate tubes several times to mix.
Place tubes in an OmniBeadRuptor, secure them, and close the lid. Run the BeadRuptor at 2.9 m/s for 00:10:00
Note
Make sure the Power Bead tubes are secure, but do not close the securing lid too tightly, 2.9 m/s is the maximum, otherwise the tubes may break.
Centrifuge at 9400 x g for 00:00:30 at Room temperature
Transfer 600 µL supernatant to the 2ml collection tube (provided in the kit), but leave the gauze in the bead tube.
Be careful not to transfer any beads or dust from the beads.
Save power bead tubes at -20 °C as backup.
Note
We save this as backup until confirmation that the extraction worked. If extraction was successful, this can be discarded.
Add 200 µL C3 solution and vortex briefly
Incubate the tubes at 4 °C for 00:05:00 on the pre-cooled rack (4 °C )
Centrifuge the tubes at 9400 x g for 00:01:00 at Room temperature
Transfer 750 µL of the supernatant to a clean 2ml collection tube (provided in the kit)
Store remaining supernatant at -20 °C
Note
We save this as backup until confirmation that the extraction worked. If extraction was successful, this can be discarded.
Gently shake C4 Solution to mix
Add 1.2 mL C4 Solution to supernatant and vortex for 00:00:05
Note
The solution should not exceed the rim of the tube.
Bind the DNA on the membrane of spin filter unit:
Load approximately 675 µL into spin filter unit
Centrifuge 00:01:00 at 9400 x g at Room temperature
Discard flow through
Repeat until the entire solution has been passed through the spin filter unit (2-3 times) go to step #21.1
Load 500 µL C5 Solution into spin filter unit
Centrifuge 00:01:00 9400 x g at Room temperature
Discard flow through
Dry spin 00:01:00 9400 x g atRoom temperature
Elution
Elution
Place the spin filter unit into a clean 2mL collection tube (provided in the kit), or if preferred, a 1.5 mL LoBind tube
Pipette 100 µL C6 Solution (pre-warmed to 37 °C ) into center of the membrane in the spin filter unit and incubate for 00:01:00
Centrifuge 9400 x g for 00:00:30 at Room temperature
Remove spin filter unit and quantify the DNA (3 µL ) with the Qubit ds BR Kit following the manufacturer's protocol
Store the eluate at-20 °C