TITLE: DNA extraction from concrete
AUTHORS: Anders  Kiledal, Julia A Maresca

[DESCRIPTION]
This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. 

Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

[BEFORE_START]
Prepare solutions
Sterilize work area
Clean rock grinder

[GUIDELINES]
Concrete is a very low biomass system, and thus susceptible to laboratory and reagent contamination. A negative control sample -- either extracting DNA with no powder, or using sterilized glass beads as the "sample" -- is strongly recommended. We also strongly recommend doing as much of this as possible inside a PCR or laminar flow hood to minimize laboratory contamination. 

[STEPS]
SECTION: Sample Pre-Treatment (Day 1)
6. In a 50 mL conical tube, place 10 g pulverized concrete, 5 mL 0.5 M EDTA, 150 µL Proteinase K (20 mg/mL), 138 µL 20% SDS, and 0.2 mL glacial acetic acid. Incubate at 55°C overnight with gentle rotation. 
SECTION: DNA Extraction (Days 2 and 3)
8. Vortex at maximum speed on a platform vortexer for 10 min (this is similar to bead beating, but using the material itself instead of beads).
SECTION: DNA Extraction (Days 2 and 3)
9. Centrifuge 3 min, 5000 RPM
SECTION: DNA Extraction (Days 2 and 3)
10. Transfer supernatant to new 50 mL conical tube
SECTION: DNA Extraction (Days 2 and 3)
11. Add 30 mL QGmod. If solution is purple (this indicates that the pH > 7.5), adjust pH with acetic acid until solution is yellow (neutral or acidic). Add 125 µl silica suspension (well mixed) and 5 µl yeast RNA (1 mg/mL, acts as a carrier molecule). Mix well and allow to bind overnight at room temperature with gentle rotation.
SECTION: DNA Extraction (Days 2 and 3)
12. Centrifuge 5 min at 5000 RPM to pellet silica.
SECTION: DNA Extraction (Days 2 and 3)
13. Decant supernatant (pour off, centrifuge the tube briefly, and carefully pipette off what remains to prevent carryover of Buffer QG. Discard supernatant.)
SECTION: DNA Extraction (Days 2 and 3)
14. Resuspend pellet in 10 mL 80% EtOH. (To prevent clogging the pipet tips, wait ~5 min after adding ethanol before trying to resuspend. It may be necessary to scrape the tube over a tube rack or bang it on the bench to resuspend the silica particles. The solution can also be stirred with the tip. Try to break up any large clumps before attempting to draw the solution into the pipet tip.)
SECTION: DNA Extraction (Days 2 and 3)
15. Centrifuge 30 min, 5000 RPM, 4°C
SECTION: DNA Extraction (Days 2 and 3)
16. Decant supernatant (pour off, pulse in centrifuge, carefully pipette off what remains)
SECTION: DNA Extraction (Days 2 and 3)
17. Resuspend silica pellet in 1 mL 80% ethanol and transfer solution to 1.5 mL microcentrifuge tube
SECTION: DNA Extraction (Days 2 and 3)
18. Centrifuge 3 min at 13000 RPM
SECTION: DNA Extraction (Days 2 and 3)
19. Decant or pipette off supernatant
SECTION: DNA Extraction (Days 2 and 3)
20. Air dry silica with bound DNA. This usually takes ~1 hour at room temperature in a PCR hood.
SECTION: DNA Extraction (Days 2 and 3)
21. Resuspend in 50 µL 10 mM Tris, pre-warmed to 60°C, then elute for 5 min at 60°C with gentle rotation. Centrifuge 3 min at 13000 RPM and transfer supernatant (with DNA) to fresh tube. Repeat and combine eluates.
SECTION: DNA Extraction (Days 2 and 3)
22. A minuscule amount of silica may remain but should not interfere with downstream applications. Any remaining silica can be pelleted to reduce future transfer.
SECTION: DNA Extraction (Days 2 and 3)
23. Quantify with Qubit and store at -20°C.
SECTION: Sample Grinding
1. Wash sample holder and puck of the ring and puck mill with soap and hot water. Rinse thoroughly and dry.
SECTION: Sample Grinding
2. Rinse sample holder and puck 2x with 95% ethanol.
SECTION: Sample Grinding
3. Sterilize sample holder and puck under germicidal UV light for 40 min.
SECTION: Sample Grinding
4. Roughly break ~10 g concrete with a hammer into pieces that are small enough to fit into a ring and puck mill or equivalent grinder
SECTION: Sample Grinding
5. Grind samples to powder (usually requires 1-5 minutes of grinding). The ground powder can be stored at -20°C (or -80°C) prior to DNA extraction.
SECTION: Sample Pre-Treatment (Day 1)
26. Some samples absorb a large volume of the pre-treatment solution, leaving very little supernatant. If this happens, add 5-10 mL of sterile water before the next step.  
SECTION: DNA Extraction (Days 2 and 3)
7. Some samples may absorb a large volume of the pre-treatment solution, leaving very little supernatant. If this happens, add 5-10 mL of sterile water before vortexing.