PMC:3514855 / 4013-6894
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3514855","sourcedb":"PMC","sourceid":"3514855","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3514855","text":"2.1. Origin of strain, isolation of DNA and PCR analysis\nSynechocystis sp. PCC 6803 substrains ‘Moscow’ here called ‘PCC-M, Kazusa (GT-Kazusa) and Vermaas’ (GT-V) were cultivated by Prof. Annegret Wilde (University of Freiburg, Germany) and maintained as frozen stocks. The ‘PCC-M’ substrain was originally obtained from the laboratory of S. Shestakov (Moscow State University) in 1993 and over the years carefully propagated for motile colonies. The ‘GT-V’ strain originates from the laboratory of W. Vermaas (Arizona State University). Genomic DNA for deep sequencing analysis was isolated from 80 ml cultures harvested on a glass microfiber filter (GF/C, 47 mm i.d. Whatman) by vacuum filtration. The frozen filter was ground in a mixer mill (Dismembrator MM301, Retsch, Germany) and the powder transferred into 1 ml SET buffer on ice (25% (w/v) sucrose, 1 mM EDTA, 50 mM Tris pH 7.5). One-fourth volume of 0.5 M EDTA, 2% SDS and 1.5 mg proteinase K (Sigma) were added for cell lysis at 50°C overnight. Following phenol/chloroform extraction, one volume of 2-propanol (Roth, Germany) was added for precipitating the DNA at room temperature for 30 min. The precipitate was washed once in H2O/2-propanol 1:1 and once in 2-propanol, followed by 10 min centrifugation at 10 000 g, 4°C. The pellet was washed with 70% EtOH, dried for 10 min and re-suspended in 50 µl H2O. One microlitre of RNase A (Sigma) was added and the tube incubated at 37°C and 260 rpm overnight. RNase was removed by another round of phenolic extraction and precipitation as described above. The DNA was re-suspended in 75 µl H2O, concentration was measured photometrically and DNA quality checked on a gel (0.8% agarose).\nGenomic DNA for PCR was isolated from the cell pellet of 1 ml Synechocystis liquid culture. The pellet was washed once with a 1:10 dilution of TE buffer (10 mM Tris HCl pH 8; 1 mM EDTA) and re-suspended in 70 µl of the same buffer. Cells were broken by incubation at 98°C for 10 min. After centrifugation at 14 000 g and 4°C for 5 min, the supernatant was collected and kept on ice. Two microlitres of it were used for PCR. For PCR reactions, Phusion® DNA polymerase (Finnzymes, New England Biolabs) was used according to the manufacturer's instructions. To verify single nucleotide polymorphisms (SNPs) between the different substrains, ∼500 bp fragments containing the SNP position were amplified. PCR products were excised from an agarose gel, purified (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare) and sent for Sanger sequencing to GATC Biotech (Konstanz, Germany). For sequencing of the small plasmids, several PCR reactions were performed to get overlapping sequences and contigs were assembled using the software ContigExpress (Vector NTI Advance 11, Invitrogen). Alignments of the sequences were performed using AlignX (Vector NTI Advance 11, Invitrogen).","divisions":[{"label":"title","span":{"begin":6,"end":57}},{"label":"p","span":{"begin":58,"end":1695}}],"tracks":[]}