, 2011). In each case, the results of the real time PCR method were in excellent agreement with the respective independent method. To give a short overview, genomic DNA was used as a template in a conventional PCR reaction to amplify a fragment of about 1 kbp. A dilution series of this fragment was prepared and used for real time PCR analysis. A fragment of about 300 bp, internal to the standard fragment, was amplified. The results were used to generate a standard curve. To determine the genome copy
number, cells were lysed and a dilution series of the resulting cell extract was analyzed using real time PCR in parallel to the standards. The results allowed calculating the number of genome UK-371804 clinical trial copies in the cell extract and, in combination with the cell density of the culture, the ploidy level. The following points have to be optimized for every new species under investigation and were optimized for the three find more species of cyanobacteria used in this study: (1) the cell density has
to be quantified with a very low variance, (2) it has to be verified that culture growth is highly reproducible, (3) the method of cell disruption has to be about 100% effective yet leaving the genomic DNA intact, and (4) the real time PCR has to be truly exponential. For cyanobacteria, the method for cell disruption turned out to be the most critical point. Several standard methods (sonification, enzymatic murein digestion, ‘normal shaking’ with glass beads) could not be used, either because the efficiency of cell lysis was too low or because damage of the genomic DNA was too high. Shaking the cells in a Speedmill with 0.1 mm glass beads led to satisfactory results, lysis efficiency VAV2 was higher than 90%, and the genomic DNA was only slightly damaged (fragment sizes from 4 kbp to >20 kbp, data not shown). The amount of beads and shaking time were optimized for every species. To exemplify the results, Fig. S1 (Supporting Information) shows one typical example of a real
time PCR analysis (Fig. S1a), a standard curve (Fig. S1b), a melting point analysis, and an analytical agarose gel of the analysis fragments (Fig. S1c, d). At least three independent cultures were analyzed (and each culture was analyzed at least in triplicates), and average values and standard deviations (SD) were calculated. Synechococcus elongatus PCC 7942 grew with a doubling time of 24 h. An average growth curve of three cultures is shown in Fig. S2. The results of genome quantification of three independent cultures are summarized in Table 1. At an OD750 nm of 0.6, S. elongatus contained about four genome copies per cell and thus the species is oligoploid. This is termed ‘exponential phase’, although growth of the cultures was not truly exponential, but the OD750 nm of 0.6 was prior to the onset of the linear growth phase (compare Fig. S2).