Division 4.1 Sample preparation

Live or freshly dead (not decomposed) oysters, which can be previously frozen, are processed for DNA extraction.

Samples are processed differently according to their size:

1. (a)

   For larvae, pools of 50 mg of the whole animals (including the shell) completed with 200 μl of distilled water are crushed and centrifuged at 1 000 g for 1 minute.

2. (b)

   For spat smaller than or of 6 mm, pools of 300 mg of the whole animals (including the shell) completed with 1 200 μl of distilled water are crushed and centrifuged at 1 000 g for 1 minute.

3. (c)

   For spat between 6 and 15 mm in size, all the soft tissues of each animal are crushed individually.

4. (d)

   For animals bigger than 15 mm, pieces of gills and mantle are isolated.

DNA extraction is performed using the QIAamp® DNA Mini Kit (QIAGEN) and following the instructions for Tissue Test Protocol.

The further sample preparation is performed in the following order:

1. 1..

   Place 100 μl of supernatant for samples referred to in (a) and (b) or 10 to 50 mg of tissues for samples referred to point (c) and (d) in a 1,5 ml microcentrifuge tube and add 180 μl of Buffer ATL.

2. 2..

   Add 20 μl Proteinase K, mix by vortexing and incubate at 56 °C until the tissue is completely lysed (overnight). Vortex occasionally during incubation to disperse sample. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

3. 3..

   Add 200 μl Buffer AL to the sample, mix by pulse-vortexing for 15 s and incubate at 70 °C for 10 minutes. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

4. 4..

   Add 200 μl ethanol (96-100 %) to the sample, and mix by pulse-vortexing for 15 s. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

5. 5..

   Carefully apply the mixture from step 4 to the QIAamp Spin Column (in a 2 ml collection tube) without wetting the rim. Close the cap and centrifuge at 10 000 rpm for 1 min. Place the QIAamp Spin Column in a clean 2 ml collection tube (provided in the kit) and discard the tube containing the filtrate.

6. 6..

   Carefully open the QIAamp Spin Column and add 500 μl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 10 000 rpm for 1 min. Place the QIAamp Spin Column in a clean 2 ml collection tube (provided in the kit) and discard the collection tube containing the filtrate.

7. 7..

   Carefully open the QIAamp Spin Column and add 500 μl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (14 000 rpm) for 3 min.

8. 8..

   (Optional) Place the QIAamp Spin Column in a new 2ml collection tube (not provided in the kit) and discard the collection tube containing the filtrate. Centrifuge at full speed (14 000 rpm) for 1 min.

9. 9..

   Place the QIAamp Spin Column in a clean 1,5 ml microcentrifuge tube (not provided in the kit) and discard the collection tube containing the filtrate. Carefully open the QIAamp Spin Column and add 100 μl of distilled water. Incubate 5 minutes at room temperature and centrifuge at 10 000 rpm for 1 min.

10. 10..

    Control the quality and efficacy of the extraction (for example by measuring OD (260 nm) under spectrophotometer or after electrophoresis in agarose gel).

11. 11..

    Prepare dilution of your samples in order to have a final DNA concentration of 50-100 ng/μl.

12. 12..

    DNA solutions are kept at 4 °C until PCR analyses are performed.

Other commercial kits may be used for the DNA extractions provided they have been demonstrated to give similar results.