Genomic dna from tissue kit

Total DNA was isolated from the gastrointestinal content samples by using the Genomic DNA from tissue Kit (Macherey-Nagel, Germany), according to the manufacturer’s instructions.

To confirm integration, genomic DNA was isolated using the Genomic DNA from Tissue Kit (Macherey–Nagel, Düren, Germany) according to the manufacturer’s recommendations for difficult-to-lyse bacteria, with one exception: after treatment with lysozyme (20 mg/ml) in lysis buffer (20 mM Tris/HCl, 2 mM EDTA, 1% Triton X-100, pH 8.0) the cells were resuspended in fresh lysis buffer (without lysozyme) before adding proteinase K. From the isolated genomic DNA 500 ng was digested with different restriction enzymes (1 µl of an appropriate restriction enzyme, e.g., AseI, BsrGI, HindIII, or PstI (New England Biolabs) in 30 µl buffer for 6 h) and 5 µl of the heat-inactivated reaction mixture was used for ligation in a reaction volume of 20 µl containing one unit of T4 DNA ligase (Thermo Fisher Scientific) at 15 °C overnight. The products were amplified from 1 µl of a 1:20 dilution using Herculase II DNA polymerase and primers iPCR_mlsR_for01 and iPCR_mlsR_rev01 for PCR (initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 3 min 30 s, then a final elongation step at 72 °C for 5 min before holding at 4 °C). The PCR products were verified by in-house sequencing using an ABI 3730 DNA Analyser (Applied Biosystems, Thermo Fisher Scientific).

Genomic DNA was purified from homogenized oyster powders using the Macherey-Nagel genomic DNA-from-tissue kit (catalog number 740952.250) following the manufacturer’s instructions, with an additional step of RNase A treatment (Macherey-Nagel; catalog number 740505). The quality and quantity of genomic DNA were estimated on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Total DNA was resuspended to a final concentration of 20 ng μl⁻¹ prior to qPCR. Total RNA was extracted from oyster pool powders (10 mg) homogenized in 500 μl of Tri-Reagent (Invitrogen) by vortexing for 2 h at 4°C. Prior to extraction, insoluble materials were removed by centrifugation at 12,000 × g for 10 min at 4°C, and supernatant was incubated with 100 μl of chloroform at room temperature for 3 min. After centrifugation at 12,000 × g for 15 min at 4°C, total RNA in the aqueous phase was extracted using the Direct-Zol RNA miniprep kit (Zymo Research; reference no. R2052) according to the manufacturer’s protocol. Quantity and purity of total RNAs were checked using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and capillary electrophoresis (Agilent BioAnalyzer 2100).