RNA Extraction and Synthesis of cDNA

1. RNA STAT-60 (TEL-TEST"B", Friendswood, TX).

2 Chloroform.

3. Isopropanol.

5. Nuclease-free water.

6. Electrophoresis-grade agarose (Bio-Rad Laboratories, San Francisco, CA).

7. 10X TBE buffer, 0.89 M Tris base, 0.89 M boric acid, 0.02 M ethylene diamino tetraacetate, disodium salt.

8. 10 mg/mL ethidium bromide (see Note 1).

9. DNAse I (RNase-free) (Boehringer Mannheim, Indianapolis, IN).

10. Human placental ribonuclease inhibitor: RNA guard (Pharmacia, Milwaukee, WI).

11. 2X Proteinase K (PK) buffer; 200 mM Tris-HCl, pH 8.0, 350 mM NaCl (5 M stock), 25 mM EDTA (0.5 M stock), 1% SDS (20% stock), 10 mg/mL PK (Boehringer). Store at -20°C.

12. Phenol-SEVAG reagent: a mixture of 25 parts of ultrapure buffer-equilibrated recrystallized phenol, 24 parts of chloroform and 1 part of isoamylalcohol (see Note 2).

13. 10X First-strand buffer; 100 mM Tris-HCl, pH 8.3 at 37°C, 40 mM MgCl2 500 mM KCl.

14. 100 mM deoxynucleotide triphosphate (dNTP) stocks (Pharmacia).

15. Cloned MuTLV reverse trancriptase (Stratagene, San Diego, CA) (see Note 3).

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