They were grown phototrophically at a fluence rate of 10–60 photons m−2 s−1 (Osram daylight lamp LUMILUX de Lux L18W/954; Osram, Munich, Germany) with constant illumination. Growth was monitored spectroscopically at 750 nm. Cultures for genome copy Selleck Kinase Inhibitor Library number determination were inoculated from precultures in the linear growth
phase and grown to the respective optical densities (see text and tables). At the times of harvest, the cultures were checked microscopically to detect possible aggregation, which was not observed, and to determine cell densities using a Neubauer counting chamber. The cells of 40 mL culture were harvested by centrifugation (3200 g, 30 min, room temperature). The supernatant was checked microscopically to verify that it was free of cells. The pellet was suspended in 2 mL distilled water. The cell density was determined microscopically using a Neubauer counting chamber. 0.5 mL of the cell suspension was mixed with either 0.75 g (Synechocystis PCC 6803) or 1 g (S. elongatus PCC 7942 and Synechococcus sp. WH7803) zirconia/silica beads (0.1 mm; Roth, Karlsruhe, Germany) in a 2 mL screw cup (Sarstedt, Nümbrecht, Germany). Cells were disrupted by shaking for 1.5 min
(Synechocystis PCC 6803) or 2 min (S. elongatus PCC 7942 and Synechococcus sp. WH7803) in a Speedmill P12 (Analytik Jena, Jena, Germany). The cell density was determined again, and the values before and after cell disruption were used to calculate the efficiency Liproxstatin-1 concentration of cell disruption. Cell debris was removed by centrifugation (15 000 g, 20 min room temperature). 0.3 mL of the supernatant was used as cytoplasmic extract for further analysis. The integrity of genomic DNA was checked using analytical agarose electrophoresis. The extract was dialyzed against distilled water, and volumes prior and after dialysis were used to calculate the dilution. To determine genome copy numbers, a real time PCR approach was applied (Breuert et al., 2006; an overview is given in Figure 1 of Pecoraro et al., almost 2011). For each species, a fragment of about 1 kbp was amplified using standard PCRs with isolated genomic DNA as template. The primers are summarized
in Table S1 (Supporting Information). The fragments were purified using preparative agarose gel electrophoresis and the AxyPrepDNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The DNA mass concentrations were determined photometrically, and the concentrations of DNA molecules were calculated using the molecular weights computed with ‘oligo calc’ (www.basic.northwestern.edu/biotools). For each standard fragment, a dilution series was generated and used for real time PCR analysis in parallel with the dilution series of the respective cell extract. The ‘analysis fragments’ were 300–400 bp, and exact sizes and primers are summarized in Table S1. The real time PCR analyses were performed as previously described (Breuert et al., 2006), but without glycerol addition.