Selected article for: "final concentration and room temperature"
Author: Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.
Title: Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection
  • Document date: 2015_11_26
    • ID: 1rrm4sqa_55
    Snippet: Afterwards, medium change cells were incubated for an additional 3 h and thereafter subjected to trypsinization. After washing of the detached control cells and the floater population (at 1 ¢ 10 6 cells per sample) with PBS, the samples were treated with lysis buffer (100 mM Tris pH 8.0, 20 mM EDTA, 0.8% SDS) for 20 min at room temperature. Thereafter, cells were incubated with 10 µL RNAse A solution (500 U/mL) for 2 h at 37 ¥ C and afterwards.....
    Document: Afterwards, medium change cells were incubated for an additional 3 h and thereafter subjected to trypsinization. After washing of the detached control cells and the floater population (at 1 ¢ 10 6 cells per sample) with PBS, the samples were treated with lysis buffer (100 mM Tris pH 8.0, 20 mM EDTA, 0.8% SDS) for 20 min at room temperature. Thereafter, cells were incubated with 10 µL RNAse A solution (500 U/mL) for 2 h at 37 ¥ C and afterwards digested with 10 µL proteinase K (20 mg/mL) overnight at 50 ¥ C. After the addition of NaCl and PEG-800 at a final concentration of 1 M and 2.5%, respectively, samples were incubated on ice for 10 min and centrifuged at 4 ¥ C for 10 min at 16,000¢ g. The resulting supernatant was collected before sodium acetate and ethanol were added at 1/10 and twofold volumes, respectively. Incubation at ¡80 ¥ C for 1 h resulted in DNA precipitation. After centrifugation at 16,000¢ g at 4 ¥ C the supernatant was discarded and the remaining pellet was washed with ice-cold 70% (v/v) ethanol and dissolved in 40 µL ddH2O. The DNA was analyzed by 1.5% standard agarose gel electrophoresis at 60 V for 30-60 min.

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