Reaktion #451011

Reaktionsgleichung

ATP

phosphocreatine

creatine

GTP

CaCl2

MgCl2

SDS

EDTA

Tris-HCl

→

guanidinium phenol chloroform

Lösungsmittel

ammonium sulfate

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeNuclei were isolated from suspension cultured cells
2. 2
   workup.ADDITIONcontaining 15 ml
3. 3
   workup.ADDITIONthe reaction mix
4. 4
   workup.WAITincubated at 26° C. for 5 minutes
5. 5
   workup.ADDITIONwas added
6. 6
   workup.WAITthe reaction was continued at 37° C. for 30 minutes

Vorschrift

Nuclei were isolated from suspension cultured cells according to the procedure of Lawton and Lamb, Mol. Cell Biol. 7, 335-341 (1987). Isolated nuclei were incubated in 0.1M ammonium sulfate, 4 mM MgCl2, 0.3 mM phosphocreatine, 0.15 mg/ml creatine phosphokinase, 1 U/ml RNasin (Promega), 0.5 mL each ATP, CTP, GTP, and 2 mCi/ml 32P-UTP (3000 Ci/mmol). The standard reaction volume was 30 ml containing 15 ml nuclei. The reaction mixture was incubated 30 minutes at 30° C. Three units RQ1 DNAse (Promega) and 2 ml 20 mM CaCl2 were added and the reaction mix incubated at 26° C. for 5 minutes. A mixture containing 1.5 ml proteinase K (2 mg/ml), 9 ml 10×SET (1×SET is 0.5% SDS, 5 mM EDTA, 10 mM Tris-HCl pH 7.4) and 50 mg yeast tRNA was added and the reaction was continued at 37° C. for 30 minutes. Labelled RNA was isolated by acid guanidinium/phenol/chloroform extraction. The assay consistently yielded 2-5×106CPMs.

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