![]() ![]() Normalized C圓/Atto635 or C圓/Cy5 ratios for individual blocks were averaged, representing final C圓/Atto635 or C圓/Cy5 ratios. coli tRNATyr2, Yeast tRNAPhe3) for individual blocks.Ĥ. median C圓/Atto635 ratios of the three tRNA standards E. C圓/Atto635 ratios were normalized to C圓/Atto635 values of tRNA standars (e.g. DNA-free Taq Polymerases and Mastermixes. The main activities are divided into these business areas: DNA/RNA synthesis - DNA/RNA analysis and sequencing - Contract research/outsourcing. Ratios of C圓/Atto635 fluorescence signals were calculated for individual spots based on median fluorescence signal (background fluorescence subtraction).ģ. DNA-free PCR Reagents for Sensitive Analyses in Molecular Microbiology. Microsynth (founded in 1989) is a leading European company in nucleic acid synthesis & analysis. TIF-files of scanned arrays were analyzed using the GenPix Pro 7 (Molecular Devices) software.Ģ. HBE cells were isolated from explanted cystic fibrosis lungs and further cultivated.ġ. Arrays were dried and storred in the dark at 4☌.Īrrays were scanned with a GenPIX 4200A (Molecular Devices) instrument.ġ indepent biological experiment was performed. oligo (TSO, IDT) and 1 L of 10 M barcoded oligo-dT (BU3, Microsynth) in a. Microarrays were washed once in 2× SSC/0.1% SDS (50☌), once in 1× SSC/0.1% SDS (42☌) and then three times in 0.1× SSC (42☌).ģ. Nextera DNA kit versus Tn5 enzymes that were produced in-house following the. Fluorescently labeled tRNAs were hybridized on the microarrays for 16 h at 60☌ in a Hyb4 microarray hybridization system (Digilab) supplemented with polyA (0.17 mg/ml) and salmon sperm DNA (0.17 mg/ml).Ģ. C圓, Cy5 or Atto653 containing oligonucleotides (Microsynth) were ligated to tRNA 3'-CCA ends using 20 U/μl T4 DNA ligase (New England Biolabs) for 16h at 16☌ in the presence of 15% DMSO (Sigma-Aldrich).ġ. Total RNA containing tRNA standards was treated for 45 min at 37☌ with 100 mM Tris-HCl (pH 9) for tRNA deacetylation.ģ. coli tRNATyr2, Yeast tRNAPhe3) were added to 2 µg extracted total RNA prior to deacetylation.Ģ. Equal amounts of in vitro synthesized tRNA standards (16 nM each E. Total RNA was extracted using TriReagent (Sigma-Aldrich) according to manufacturer's protocol.ġ. Primary HBE cells were cultivated as air liquide interface (ALI) cultures on transwell filters in ALI medium until confluency at 37☌ and 5% CO2.Ĭell were grown independently for each biological experiment and checked frequently for mycoplasma contamination. GEO help: Mouse over screen elements for information.Ĭell type: CF patients derived bronchial epithelial cellsĬells were cultivated at 37☌ and 5% CO2 in MEM (Biochrome) (HEK, CFBE41o-) or DMEM (PAN Biotech) (HeLa) medium supplemented with 10% FCS (PAN Biotech) and 2 mM L-glutamine (Gibco) until 70 to 80% confluency.
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