TITLE: DNA Extraction from Modern Dental Plaque on Gauze
AUTHORS: Franziska   Aron, Irina  Velsko

[DESCRIPTION]
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. 

[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.


[STEPS]
SECTION: Preparation Day 1
1. Place a 1.5ml/2ml tube rack at 4 °C for the incubation steps on the final days

SECTION: Preparation Day 1
2. Label for each sample one 2ml-Eppendorf Safe-Lock DNA LoBind-Tube
 and one 1.5ml-Eppendorf Safe-Lock DNA LoBind-Tube 
SECTION: Preparation for Final Day
4. Retrieve the spin filter columns (provided in the kit) from fridge to warm them up to -20 Room temperature for use at step 25
 

SECTION: Preparation for Final Day
5. Label for each sample one Power Bead tube, one spin filter column and three 2ml collection tubes (all provided in the kit).
 

SECTION: DNA_Clean_up
9. Invert the PowerSoil bead tubes to mix and fully submerge gauze with plaque. 
SECTION: DNA_Clean_up
10. Add  60 µL  C1 solution, invert several times to mix.



SECTION: DNA_Clean_up
12. Place tubes in an OmniBeadRuptor, secure them, and close the lid. Run the BeadRuptor at 2.9 m/s for 10 min

 

SECTION: DNA_Clean_up
13. Centrifuge at 9400 x g for 30 s at Room temperature 





SECTION: DNA_Clean_up
14. 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. 


SECTION: DNA_Clean_up
14.1. Save power bead tubes at -20 °C as  backup. 

 

SECTION: DNA_Clean_up
15. Add 200 µL  C3 solution and vortex briefly

SECTION: DNA_Clean_up
16. Incubate the tubes at 4 °C for 5 min on the pre-cooled rack (4 °C )


SECTION: DNA_Clean_up
17. Centrifuge the tubes at 9400 x g for 1 min at 4 Room temperature  

SECTION: DNA_Clean_up
18. Transfer 750 µL of the supernatant to a clean 2ml collection tube (provided in the kit)

SECTION: DNA_Clean_up
18.1. Store remaining supernatant at -20 °C  
 


SECTION: DNA_Clean_up
19. Gently shake C4 Solution to mix
SECTION: DNA_Clean_up
20. Add 1.2 mL C4 Solution to supernatant and vortex for 5 s

 

SECTION: DNA_Clean_up
21. Bind the DNA on the membrane of spin filter unit: 

SECTION: DNA_Clean_up
21.1. Load approximately 675 µL into spin filter unit



SECTION: DNA_Clean_up
21.2.  Centrifuge  1 min at 9400 x g at 4 Room temperature
SECTION: DNA_Clean_up
21.3. Discard flow through
SECTION: DNA_Clean_up
22. Repeat until the entire solution has been passed through the spin filter unit (2-3 times)   

SECTION: DNA_Clean_up
23. Load 500 µL C5 Solution into spin filter unit

SECTION: DNA_Clean_up
24. Centrifuge 1 min 9400 x g at -20 Room temperature

SECTION: DNA_Clean_up
25. Discard flow through
SECTION: DNA_Clean_up
26. Dry spin  1 min 9400 x g at-20 Room temperature

SECTION: Elution
27. Place the spin filter unit into a clean 2mL collection tube (provided in the kit), or if preferred, a 1.5 mL LoBind tube


SECTION: Elution
28. Pipette 100 µL C6 Solution (pre-warmed to 37 °C) into center of the membrane in the spin filter unit and incubate for 1 min

SECTION: Elution
29. Centrifuge 9400 x g for 30 s at -20 Room temperature

SECTION: Elution
30. Remove spin filter unit and quantify the DNA (3 µL) with the Qubit ds BR Kit following the manufacturer's protocol

SECTION: Elution
30.1.  Store the eluate at-20 °C


SECTION: Sample Preparation
6. 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. 
 

SECTION: DNA_Clean_up
11. Add 25 µLproteinase K (10 mg/mL), and rotate tubes several times to mix. 

SECTION: Preparation Day 1
3. Place solution C6 in an incubator to pre-heat it to 37 °C. 

SECTION: Sample Preparation
8. 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.
SECTION: Sample Preparation
7. 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.