Materials see Note 1

1. Lysis buffer: 10mMHEPES,pH7.9, lOmMNaCl, 3 mMMgCl2,0.5% NP40, and 50 U/mL human placental RNase inhibitor (RNasin, Amersham, UK).

2. Storage buffer: 50 mM HEPES, pH 7.9, 40% (v/v) glycerol, 5 mM MgCl2, 0.25 mMEDTA, and 500 U/mL RNAsin.

3. 10X Run-on buffer. 1.5MNaCl, 25 mMMgCl2, 50 mMMg acetate, 10 mM MnCl2, 20 mM DTT, 1 25 mM EDTA, 5 mM ATP (Pharmacia, Uppsala, Sweden), 5 mM GTP (Pharmacia), 5 mM CTP (Pharmacia), 20 mM creatine phosphate, 30 U/mL creatine phosphokinase (Boehringer Mannheim, Mannheim, Germany), and 5 mg/mL heparin.

4. Nucleoside 5' diphosphatekinase (Boehringer Mannheim).

5. a-32P-labeled UTP (3000 Ci/mmol; Amersham) (see Note 2).

6. DEPC (diethylpyrocarbonate)-treated water (see Note 1). This is prepared by adding 0.1% (v/v) DEPC to distilled, deionized, or reverse-osmosis purified water. Following incubation at room temperature for 3 h, the mixture is autoclaved.

7 Vanadylribonucleoside complex (VRC; Gibco-BRL, Gaithersburg, MD)

8. RNase-free pancreatic DNase I

9. 10X Proteinase K(PK) buffer: 10 mMTris-HCl,pH 7.4,50 mMEDTA, 10% (w/v) SDS, and 1.6 mg/mL PK (Sigma, St. Louis, MO, or BRL).

10 Tris-buffered phenol/chloroform/isoamyl alcohol (24:24:1): To prepare Tris-buffered phenol, 100 g of molecular-biology-grade phenol (BRL) is melted at 65°C and equilibrated overnight with 100 mL Tns-HCl, pH 7.4, 24 mL 0.5MEDTA, 7.5 mL 4MNaOH, and 243.5 mL DEPC-treated water. Following the removal of most of the upper aqueous phase, 8-hydroxy-quinoline (100 mg) and P-mercaptoethanol (200 |oL) are then added, and the phenol is stored frozen.

11 DEPC-treated 0.3M Na acetate, pH 5.5 {see Note 1): DEPC is added to the solution to 0.1% (v/v). Following incubation at room temperature for 3 h, the solution is autoclaved.

12. DEPC-treated 0 2M NaOH (see Note 1) DEPC is added to the solution to 0.1 % (v/v). Following incubation at room temperature for 3 h, the solution is autoclaved. 13 2.4M HEPES.

14. Sephadex G-50 slurry: This is prepared by swelling G-50 powder in water followed by autoclaving.

15. Disposable 1-mL syringes.

16 Autoclaved glass wool.

17. Wash buffer A: 50 mMNa phosphate buffer, pH 6.8, and 0.1% (w/v) SDS.

18. Wash buffer B: 50 mM Na phosphate buffer, pH 6.8, and 1% (w/v) SDS.

19. 2X SSPE: 360 mM NaCl, 20 mM Na phosphate buffer, pH 6.8, and 1 mM EDTA.

20. DNase-free RNase A (10 mg/mL): This is made by resuspending the RNase A dry powder in water. Following incubation in a boiling water bath for 10 min and quenching on ice, the solution is aliquoted and frozen at -20°C until required.

22. 5M NH4 acetate.

23. Hybond-N (Amersham) nylon hybridization membrane (see Note 3).

24. Slot-blot apparatus linked to a vacuum line.

25. Ultraviolet light (312 nm) transilluminator.

26. Hybridization buffer: 500 mM Na phosphate buffer, pH 6.8,1% (w/v) SDS, 1 mM EDTA, and 15% (v/v) formamide (Fluka, Buchs, Switzerland) {see Note 4).

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