TITLE: Coral tissue and skeleton DNA extraction
AUTHORS: Molly Moynihan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This extraction protocol is based on the Qiagen DNeasy PowerBiofilm kit with modifications from Sunagawa et al. 2010.</div><div class = "text-block">Sunagawa, S., Woodley, C. M. & Medina, M. Threatened Corals Provide Underexplored Microbial Habitats. PLoS ONE 5, e9554 (2010).</div></div>

[STEPS]
?. [Preparation]
If needed, prepare lysozyme by dissolving  of lysozyme powder (≥ 40,000 units/mg)  per  of nuclease free water. Multiple aliquots can be prepared and stored at .
12.5 mg
1 mL
-20 °C
?. [Preparation]
Thaw samples and lysozyme (if frozen).
?. [Preparation]
Clean surfaces and pipettes with 10% bleach and 70% ethanol.
?. [Preparation]
Warm solution solution MBL at  for .
65 °C
?. [Preparation]
Pre-label tubes.
?. [Extraction]
Add  to each bead tube.
[warm MBL solution]
?. [Extraction]
Add  to each bead tube. Vortex briefly.
[solution FB]
?. [Extraction]
Add  (see Step 1) to the bead sample mixture. Incuate at  for .
[lysozyme (12.5mg/ml)]
0 Room temperature
?. [Extraction]
Add  to each sample and incubate at  for .
[proteinase-K (20mg/ml)]
65 °C
?. [Extraction]
Bead beat the sample with a Vortex Adapter for .
?. [Extraction]
Centrifuge the tubes at .
Centrifuge: 13000 34, 1 min
?. [Extraction]
Add   and vortex briefly to mix. Incubate at  for
[solution IRS]
4 °C
?. [Extraction]
Centrifuge the tubes at .
Centrifuge: 13000 34, 1 min
?. [Extraction]
Avoiding the pellet, transfer all of the supernatant to a 2ml collection tube.
?. [Extraction]
Transfer the supernatant to a clean 2ml collection tube.
?. [Extraction]
Add  and vortex briefly.  (Note: if solution MR has precipitated, warm at 55°C for 5-10 minutes)
[solution MR]
?. [Extraction]
Load  onto an MB spin column and centrifuge at  . Discard the flow-through and repeat until all the supernatant has been processed.
[supernatant]
Centrifuge: 13000 34, 1 min
?. [Extraction]
Place the MB Spin Column into a clean 2ml Collection Tube.
?. [Extraction]
Shake Solution PW. Add  and centrifuge at  .
[solution PW]
Centrifuge: 13000 34, 1 min
?. [Extraction]
Discard the flow-through and add  and centrifuge at .
[ethanol]
Centrifuge: 13000 34, 1 min
?. [Extraction]
Discard the flow-through and centrifuge again at  to ensure all ethanol is removed from the filter.
Centrifuge: 13000 34, 2 min
?. [Extraction]
Place the MB Spin Column basket into a clean 2ml collection tube.
?. [Extraction]
Add  to  of nuclease free water to the center of the white filter membrane, depending on expected yield and desired concentration. Make sure entire membrane is wet. Incubate at room temperature for  .
60 µl
100 µl
?. [Extraction]
Centrifuge at  and discard the MB Spin Column.
Centrifuge: 13000 34, 1 min
?. [Extraction]
Proceed to DNA quantification and dilute (if needed) with nuclease-free water.Store at  for short term storage (e.g. to be used within the same week) or for long term storage.
-20 °C
-80 °C
?. [Extraction]
Add between   to   of airbrushed coral tissue or  -    of pulverized skeleton to each bead tube.  The optimal amount of starting material may vary for each sample, depending on how much PBS was used to airbrush sample, the amount of material of the skeleton's endolithic community, and the coral species. Too much starting material can result in low yields, particularly for coral species with thick tissue.
200 µl
400 µl
200
500