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Typus
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Bearbeiter
Graf Isolan
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Untersuchte Arbeit:
Seite: 45, Zeilen: 1-12
Quelle: Novosyadlyy 2004
Seite(n): 89, Zeilen: 1-13
[Radiolabelled probe (1,500,000-3,000,000 cpm/ml) was mixed with a double volume] of fish sperm DNA and denatured at 95°C for 5 min. After cooling down on ice, the DNA probe was applied into QuikHyb® solution inside the hybridization tube. The tube was placed back in the hybridization oven, and hybridization was carried out at 68°C for 2 hours. After hybridization, the membrane was washed once in 2x SSC/0.1% SDS for 10 min at RT, then twice in 0.1x SSC/0.1% SDS for 15 min at 430C (IGF-IR, IGF-II/M6-PR cDNA), 55 0C (IGF-I, cDNA) or 600C (IGFBP-2, IGFBP-3, cDNA) and, finally, twice in 2x SSC/0.1% SDS for 10 min at RT. To visualize 28S rRNA, the membrane was prehybridized in QuikHyb® hybridization solution at 42°C for 2 hour followed by overnight hybridization at 42°C with the labelled oligonucleotide specific for 28S rRNA and washed three times in 2x SSC/0.1% SDS for 10 min at 370C. After washing, membranes were wrapped in a saran wrap, placed in X-ray film cassette and autoradiographed during various exposure times. [Radiolabelled probe (1,500,000-] 3,000,000 cpm/ml) was mixed with a double volume of fish sperm DNA and denatured at 95°C for 5 min. After cooling down on ice, the DNA probe was applied into QuikHyb® solution inside the hybridization tube. The tube was placed back in the hybridization oven, and hybridization was carried out at 68°C for 2 h. After hybridization, the membrane was washed once in 2x SSC/0.1% SDS for 10 min at RT, then twice in 0.1x SSC/0.1% SDS for 15 min at 430C (IGF-IR, IGF-II/M6-PR, PDGFRβ cDNA), 55 0C (IGF-I, IGF-II, PDGFRα cDNA) or 600C (IGFBP-2, IGFBP-3, α2(I) chain of type I procollagen cDNA) and, finally, twice in 2x SSC/0.1% SDS for 10 min at RT. To visualize 28S rRNA, the membrane was prehybridized in QuikHyb® hybridization solution at 42°C for 2 hours followed by overnight hybridization at 42°C with the labelled oligonucleotide specific for 28S rRNA and washed three times in 2x SSC/0.1% SDS for 10 min at 370C. After washing, membranes were wrapped in a saran wrap, placed in X-ray film cassette and autoradiographed during various exposure times.
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