Creatine → Guanidinium phenol chloroform

HRID 363829

Reaktionsgleichung

Reaktionsgleichung von HRID 363829
[Mg+2].[Cl-:2].[Cl-:3].P(C([N:12]([C:14](=[NH:16])[NH2:15])C)C(O)=O)(O)(O)=O.O=C(CN(C(=N)N)C)O.P(OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.P(O[CH2:70][C@H]1O[C@@H](N2C3N=C(N)NC(=O)C=3N=C2)[C@H](O)[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.[Cl-:89].[Cl-].[Ca+2].CCCCCC[CH2:98][CH2:99][CH2:100][CH2:101][CH2:102][CH2:103][O:104]S([O-])(=O)=O.[Na+].C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O.C(O)C(N)(CO)CO.Cl>S([O-])([O-])(=O)=O.[NH4+].[NH4+]>[NH2:15][C:14]([NH2:16])=[NH2+:12].[C:103]1([OH:104])[CH:98]=[CH:99][CH:100]=[CH:101][CH:102]=1.[CH:70]([Cl:89])([Cl:3])[Cl:2]

Komponenten

11

Edukt 9

HCID 586CreatineHCID 59575'-AtpHCID 6049Ethylenediaminetetraacetic AcidHCID 93573Tromethamine hydrochlorideHCID 3423265Sodium Lauryl SulfateHCID 5284359Calcium ChlorideHCID 5360315Magnesium ChlorideHCID 18506526HCID 18506526, VerbindungHCID 135398633Guanosine Triphosphate

Produkt 1

HCID 131720809Guanidinium phenol chloroform

Reagenzien, Katalysatoren, Lösungsmittel 1

HCID 6097028Ammonium SulfateLösungsmittel

Versuchsvorschrift

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.

Aufarbeitung

  1. customNuclei were isolated from suspension cultured cells
  2. additioncontaining 15 ml
  3. additionthe reaction mix
  4. waitincubated at 26° C. for 5 minutes
  5. additionwas added
  6. waitthe reaction was continued at 37° C. for 30 minutes

Quellen und Nachweise

ORD-Eintrag
ord-7dcfdbcae3384611adae94e542176ee9
Quelldatensatz
uspto-grants-1999_02
Patentnummer
US05874626

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