Total DNA was prepared from Synechocystis 6803 by collecting cells from 50 ml cultures at an OD750 of 1–1.2 by centrifugation at 3,237 g for 10 min. The pellets were resuspended in 1 ml SET buffer (50 mM Tris, pH 7.5; 1 mM EDTA, 25% (w/v) sucrose). For lysis, resuspended cells were incubated in 100 mM EDTA (pH 8), 2% SDS (w/v) and 100 μg/ml proteinase K at 50°C for 16 h. DNA was extracted by phenol/chloroform/isoamylalcohol (25:24:1) extraction. DNA was precipitated from the final aqueous phase by adding 1 vol isopropanol, incubation at-20°C for at least 2 h and centrifugation at 15,871 g for 30 min at 4°C. The pellet was washed with 70% ethanol, air-dried and resuspended in 30 μl sterile Milli-Q water.
For restriction analysis, 4 μg of total DNA each were digested with FastDigest HindIII restriction endonuclease for 3 h at 37°C to ensure complete digestion. The digested DNA samples were subjected to gelelectrophoretic separation for 1 to 2 h at room temperature on ethidium bromide stained 0.8% agarose gels with 120 V. Thereafter, the gels were incubated at 70 rpm at room temperature for 30 min each in 0.25 M HCl, in denaturation solution (1.5 M NaCl, 0.5 M NaOH) and in neutralization solution (1.5 M NaCl, 0.5 M Tris pH 7.5) prior to blotting. The DNA samples were blotted onto Hybond-N+ nylon membranes (Cytiva) by capillary transfer overnight with 20x SSC (3 M NaCl, 300 mM sodium citrate pH 7) as transfer buffer. After blotting the DNA was crosslinked to the membrane with 120 mJ using a UV-Stratalinker (Stratagene).
For the generation of isotope-labelled probes, templates were amplified via PCR, using the primers P38 and P40 containing the T7 promoter sequence together with the respective primers P37 and P39 and genomic DNA from Synechocystis 6803. Transcript probes labelled with [α-32P]-UTP (3,000 Ci/mmol, 10 mCi/ml) were generated from these templates using the MAXIscript® T7 In Vitro Transcription Kit (Thermo Fisher Scientific).
Hybridization was performed overnight at 52°C in hybridization buffer followed by 10 min wash steps each in wash buffers 1, 2 and 3 (for buffers see Northern blot section) at 47°C. The signals were detected with a Phosphor Imaging Screen (Bio-Rad) and the GE Healthcare Typhoon FLA 9500 imaging system.
For restriction analysis, 4 μg of total DNA each were digested with FastDigest HindIII restriction endonuclease for 3 h at 37°C to ensure complete digestion. The digested DNA samples were subjected to gelelectrophoretic separation for 1 to 2 h at room temperature on ethidium bromide stained 0.8% agarose gels with 120 V. Thereafter, the gels were incubated at 70 rpm at room temperature for 30 min each in 0.25 M HCl, in denaturation solution (1.5 M NaCl, 0.5 M NaOH) and in neutralization solution (1.5 M NaCl, 0.5 M Tris pH 7.5) prior to blotting. The DNA samples were blotted onto Hybond-N+ nylon membranes (Cytiva) by capillary transfer overnight with 20x SSC (3 M NaCl, 300 mM sodium citrate pH 7) as transfer buffer. After blotting the DNA was crosslinked to the membrane with 120 mJ using a UV-Stratalinker (Stratagene).
For the generation of isotope-labelled probes, templates were amplified via PCR, using the primers P38 and P40 containing the T7 promoter sequence together with the respective primers P37 and P39 and genomic DNA from Synechocystis 6803. Transcript probes labelled with [α-32P]-UTP (3,000 Ci/mmol, 10 mCi/ml) were generated from these templates using the MAXIscript® T7 In Vitro Transcription Kit (Thermo Fisher Scientific).
Hybridization was performed overnight at 52°C in hybridization buffer followed by 10 min wash steps each in wash buffers 1, 2 and 3 (for buffers see Northern blot section) at 47°C. The signals were detected with a Phosphor Imaging Screen (Bio-Rad) and the GE Healthcare Typhoon FLA 9500 imaging system.
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