DNA Extraction from Modern Dental Plaque on Gauze
Franziska Aron, Irina Velsko
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.
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.
Steps
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
Retrieve the spin filter columns (provided in the kit) from fridge to warm them up to -20Room temperature for use at step 25
Label for each sample one Power Bead tube, one spin filter column and three 2ml collection tubes (all provided in the kit).
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.
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.
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
Invert the PowerSoil bead tubes to mix and fully submerge gauze with plaque.
Add 60µL C1 solution, invert several times to mix.
Add 25µLproteinase K (10mg/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 0h 10m 0s
Centrifuge at 9400x g,0h 0m 0s for 0h 0m 30s at Room temperature
Transfer 600µL 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.
Add 200µL C3 solution and vortex briefly
Incubate the tubes at 4°C for 0h 5m 0s on the pre-cooled rack (4°C )
Centrifuge the tubes at 9400x g,0h 0m 0s for 0h 1m 0s at 4Room temperature
Transfer 750µL of the supernatant to a clean 2ml collection tube (provided in the kit)
Store remaining supernatant at -20°C
Gently shake C4 Solution to mix
Add 1.2mL C4 Solution to supernatant and vortex for 0h 0m 5s
Bind the DNA on the membrane of spin filter unit:
Load approximately 675µL into spin filter unit
Centrifuge 0h 1m 0s at 9400x g,0h 0m 0s at 4Room temperature
Discard flow through
Repeat until the entire solution has been passed through the spin filter unit (2-3 times)
Load 500µL C5 Solution into spin filter unit
Centrifuge 0h 1m 0s 9400x g,0h 0m 0s at -20Room temperature
Discard flow through
Dry spin 0h 1m 0s 9400x g,0h 0m 0s at-20Room temperature
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 100µL C6 Solution (pre-warmed to 37°C) into center of the membrane in the spin filter unit and incubate for 0h 1m 0s
Centrifuge 9400x g,0h 0m 0s for 0h 0m 30s at -20Room temperature
Remove spin filter unit and quantify the DNA (3µL) with the Qubit ds BR Kit following the manufacturer's protocol
Store the eluate at-20°C
