FastDigest Bsh1236I

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	Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min.

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Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min.

Publication protocol

"The diversity of the dominant members of the three microbial domains was characterised by genetic profiling using terminal restriction fragment length polymorphism (T-RFLP). The 16S rRNA genes of Bacteria were amplified using primers F27* (Lane, 1991) and 926r (Liu et al.,1997), those of Archaea with A364aF (Burggraf et al.,1997) and A934bR* (Grosskopf et al., 1998), and for Fungi, the region between the SSU and the large subunit rRNA gene was amplified using ITS1f* (Gardes & Bruns, 1993) and ITS4r (White et al., 1990). Asterisks indicate fluorescence-labelled primers. PCR was conducted in a total volume of 25 lL which contained 0.025 U lL1 HotStar Taq DNA Polymerase, 19 buffer (both Qiagen,Hilden, Germany), 0.2 lM of each dNTP (Carl Roth, Karlsruhe, Germany), and 0.5 lM of each primer. The concentration of MgCl2 was 1.75 mM for amplifications targeting Bacteria, 2.75 mM for Fungi and 3 mM for Archaea. For the Fungi-specific PCR, the reaction solution also contained 0.4 mg mL1 bovine serum albumin (New England Biolabs, Ipswich). Templates for PCR consisted of 1 lL of a 100-fold dilution for Bacteria (10-fold for the coarsest fraction) or 10-fold for Archaea (undiluted for the coarsest fraction) and Fungi. The PCR started at 95 °C for
15 min, then followed 30 cycles at 94 °C for 30 s, 72 °C for 90 s for Bacteria or 60 s for Archaea and Fungi and a final extension at 72 °C for 5 min (8 min in the case of Bacteria). The annealing conditions were 40 s at 50 °C for Bacteria, 50 s at 60 °C for Archaea and 40 s at 55 °C for Fungi, respectively. To minimise variability, all PCRs were carried out in triplicates, which were afterwards unified. The size and purity of the amplicons were analysed by agarose gel electrophoresis. A total of 70 lL of the PCR products were purified with the Hi Yield Gel/PCR DNA Fragments Extraction Kit (Sud-Laborbedarf GmbH, Gaut- €
ing, Germany) according to manufacturer’s instructions and eluted with 30 lL elution buffer. The DNA concentration was determined with NanoDrop 2000c (Thermo Fischer Scientific), and then 100 ng of DNA was used for digestion with restriction endonuclease (final concentration of 0.3 U lL1 ) and the corresponding buffer in a total volume of 30 lL. Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1
volumes of 3 M sodium acetate (pH 4.6) at 20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 lL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 lL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQTM 8800;Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min. Profiles were analysed by setting the maximum bin width to 2 bp. Total peak heights were normalised to 100%. Fragments representing < 1.5% of the total peak heights were considered to be noise, and bins, which
appeared in only one sample, were outliers. Independent profiles of the tree restriction enzymes for Bacteria and Fungi were additively combined to single profiles for eachdom"

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