Three solutions were prepared in the laboratory and placed in labelled 8 mL screw cap vials (Sarstedt, Nümbrecht, Germany). The reagents and concentrations of the preservatives were as follows: TNx2, 800 mM NaCl, 20 mM Tris HCl, 1.2% of a 10% SDS solution, 200 mM EDTA, pH 8.0 adjusted (with NaOH), 30 ppm ProClin 300 (Sigma-Aldrich, St. Louis, MO, USA); GHx2, 2 M GuHCl, 200 mM Tris HCl, 6% Tween20, 1% Triton X-100, 40 mM EDTA, pH 8.0 adjusted (with NaOH), 30 ppm ProClin 300 (Sigma-Aldrich); and EtOH, 100% ethanol. Rumen sample fluid is added to the screw capped vials in ratios (sample to preservative solution) of 1:1 for TNx2 and GHx2 and 1:2 for EtOH (Table 1 ).
Composition and concentration of preservation solutions after adding rumen sample
| Solution | Function | Reagent | Final conc.1 |
|---|---|---|---|
| TNx2 | Salt | NaCl | 400 mM |
| Buffering agent | Tris | 10 mM | |
| Surfactant | SDS | 0.6% | |
| Chelating agent | EDTA | 100 mM | |
| Bacteriostat | ProClin 300 | 15 ppm | |
| GHx2 | Salt | GuHCl | 1 M |
| Buffering agent | Tris | 100 mM | |
| Surfactant | Tween20 | 3.0% | |
| Surfactant | Triton X100 | 0.5% | |
| Chelating agent | EDTA | 20 mM | |
| Bacteriostat | ProClin 300 | 15 ppm | |
| EtOH | Preservative | 100% ethanol | 66% |
1Final concentration after rumen sample fluid is added at ratios (sample to preservative solution) of 1:1 for TNx2 and GHx2 and 1:2 for EtOH