Reagents for Isolation of Total RNA

1. Guanidine thiocyanate solution (GuSCN): 4.0 M guanidine thiocyanate (BMB #100-175), 0.01 M Na-acetate, pH 7.0, 0.1 M 2-mercaptoethanol (Sigma), 0.1% (w/v) n-lauryl sarcosine (Sigma), 0.5% (v/v) antifoam C (Sigma).

2. CsCl-EDTA cushion: 6.0 M CsCl (BMB), 0.1 M EDTA, pH 7.0.

3. Phenol:chloroform:isoamyl alcohol: 25 parts ultrapure phenol (BMB #100-300), 24 parts chloroform (Fisher ACS grade, Fisher Scientific, Pittsburgh, PA), 1 part isoamyl alcohol (Fisher ACS grade). Prepared as described in ref. 9.

4. Chloroform:isoamyl alcohol: 24 parts chloroform, 1 part isoamyl alcohol, store at room temperature in a dark bottle.

5. Ethanol-sodium acetate: 0.04 M Na-acetate, 60% v/v ethanol, prepare by mixing 3 parts of 80% ethanol with 1 part of 0.15 M Na-acetate, pH 7.0.

6. Ammonium acetate for precipitations: 7.5 M NH4-acetate pH 7.0—treat with 0.2% DEPC and autoclave. Store at -20°C.

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