Each PCR from the two samples was repeated with different cycle numbers (between 20 and 37). The lowest number of cycles that gave a positive signal, that is, 26 and 28 cycles for the March and April sample, respectively, was further Abiraterone manufacturer used in order to eliminate some of the major PCR innate limitations [29, 30] and to avoid differential representation of 18S rRNA genes with low and high copy numbers.For PCR amplification of the Cyanobacterial 16S rDNAs, we used the Cyanobacteria-specific primers CYA106f (5��-CGGACGGGTGAGTAACGCGTGA-3��), CYA781r(a) (5��-GACTACTGGGGTATCTAATCCCATT-3��), and CYA781r(b) (5��-GACTACAGGGGTATCTAATCCCTTT-3��) [31]. PCR included an initial denaturation step at 94��C for 5min, which was followed by 40 cycles consisting of denaturation at 94��C for 30s, annealing at 57��C for 30s, and elongation at 72��C for 3; a final 5 min elongation step at 72��C was included.

Cycle optimization was performed as above which resulted in 26 cycles for the March sample. In April 2010, no sample was analysed for 16S rRNA gene diversity since the vast majority of the observed morphospecies was observed microscopically.The PCR products from both the Eukarya- and Cyanobacteria-specific amplifications were visualized on a 1% agarose gel under UV light, purified using the Montage purification kit (Millipore Inc, Molsheim, France). The purified PCR products were ligated into the PCR XL TOPO Vector (Invitrogen-Life Technologies, Carlsbad CA, USA) and transformed in electrocompetent Escherichia coli cells according to the manufacturer’s specifications.

For each clone library a maximum of 151 clones were sequenced, each containing an insert of ca. 1800/1600 or 680bp for the Eukarya and Cyanobacteria, Entinostat respectively. These clones were grown in liquid Luria-Bertani medium with kanamycin and their plasmids were purified using the Nucleospin Plasmid QuickPure kit (Macherey-Nagel GmbH and Co. KG, D��ren, Germany) for DNA sequencing. Sequence data were obtained by capillary electrophoresis (Macrogen Inc., Seoul, Korea) using the BigDye Terminator kit (Applied Biosystems-Life Technologies, Carlsbad, CA, USA) with the set of primers M13F (5��-GTAAAACGACGGCCAG-3��) and M13R (5��-CAGGAAACAGCTATGAC-3��). For the eukaryotic clones, intermediate sequencing was performed using the primer 1179rE (5��-CCCGTGTTGAGTCAAATT-3��) [32]. Each sequence read was approximately 850bp. For each individual clone, forward, reverse, and intermediate��for the Eukarya��reads were assembled, and then the assembled sequences were checked for chimeras. The Pintail program (http://www.bioinformatics-toolkit.