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发布于:2019-2-12 04:01:49  访问:10 次 回复:0 篇
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Foil and incubated 4? days at 42 . To observe the effects of photoreactivation
All authors read and authorized the final manuscript.Additional material More FileAccession numbers for phylogenetic analysis. Accession numbers utilized to produce data presented in Figure five. Click here for file [http://www.biomedcentral.com/content/supplementary/17461448-2-11-S1.doc]Two identical dot blots were ready on nitrocellulose filters, every single containing a set of dilutions of each DNA sample in 1 M ammonium acetate. A single blot was utilized to measure total damage (CPDs and 6-4PPs); the other was exposed to CPD photolyase and visible light to do away with CPDs and let for detection of 6-4PPs alone. The blots had been then exposed to rabbit polyclonal antiserum containing antibodies to 6-4PPs and CPDs, then to biotinylated anti-rabbit antibody followed by alkaline phosphatase-conjugated Extravidin (Sigma) and lastly to Nitro Blue tetrazolium (NBT) and 5-bromo-4-chloroindolyl phosphate (BCIP) substrate. The intensity of blue color was proportional towards the level of DNA harm within the samples and was measured using a scanning densitometer (BioRad GS-670) and in comparison to a set of standards included on every blot.Foil and incubated four? days at 42 . To observe the effects of photoreactivation on survival, two identically spotted plates have been exposed to Sylvania GroLux fluorescent light (Sylvania F40/GRO/AQ/RP) for 24 hours prior to incubation at 42 . One particular plate was wrapped in foil as a manage.Repair experiments and Immunoassays for measurement of photoproducts The repair experiments and dot-blot immunoassays for UV photoproducts had been carried out as described previously [12,15,21,41]. Log-phase cells had been harvested and irradiated with 150 J/m2 UV-C at a dose rate of 1 J/m2/sec as well as the irradiated cells were incubated aerobically at 37 to permit repair to proceed. We‘ve previously shown that, immediately after this dose of UV, there is PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25681438 no detectable DNA replication IMD-0354cost throughout a 3-hour post-UV incubation [15]. All irradiation and post-UV incubation was carried out either below yellow light illumination or within the dark. Fifty-ml samples had been harvested at timed intervals and genomic DNA extracted utilizing Promega Wizard genomic DNA kits. DNA samples in the numerous time points have been equalised by measuring fluorescence of ethidium bromide-stained DNA in agarose gels, adjusting DNA concentrations and repeating this evaluation as several times as needed until all samples were of equal concentration.and 0322, respectively. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 Sequences for Haloarcula marismortui, Natronomonas pharaonis, and Methanosphaera stadtmanae had been downloaded from NCBI (see Added file 1). Amino acid sequences had been aligned making use of CLUSTAL_X1.83 [42]. Alignments were manually inspected and edited if essential. TREEPUZZLE5.two was made use of for quartet puzzling consensus maximum likelihood phylogenetic reconstruction using the JTT amino acid substitution matrix [43].Competing interestsThe author(s) declare that they‘ve no competing interests.Authors‘ contributionsDJC and SM conceived and designed the study and cowrote the manuscript. DJC and MC constructed the mutants and carried out preliminary characterization. ER, LS and IB confirmed mutant genotypes by PCR and Southern evaluation; ER and LS performed UV survival experiments.
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