Rapid Amplification of cDNA Ends RACE

1. Primers used for 5' RACE cloning are shown in Table 1.

2. DEPC-treated dH2O (see Subheading 2.4., item 4).

3. Superscript II reverse transcriptase (200 U/mL), 0.1 M dithiothreitol (DTT) and 5X buffer: 250 M Tris-HCl (pH 8.3), 0.375 M KCl, 15 mM MgCl2 (Gibco-BRL 18064-014).

4. 1 M Sodium hydroxide.

5. 1 M Hydrochloric acid.

6. Microdialysis filters: 0.025 and 0.1 |im pore size (Millipore, Watford, UK VSWP02500 and VCWP02500).

7. TE buffer: 10 mM Tris-HCl, pH 8.0 (20°C), 1 mM EDTA, pH 8.0.

8. Terminal deoxynucleotidyl transferase (TdT): 15 U/|L and 5X reaction buffer: 0.5 M potassium cacodylate (pH 7.2), 10 mM cobalt chloride, 1 mM DTT (Gibco-BRL 18008-011).

9. dATP for tailing reaction: 2 mM dATP.

10. Restriction buffer M: 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT (Boehringer-Mannheim 1417 983).

11. dNTP mix: dNTPs each at a final concentration of 10 mM (Pharmacia, St. Albans, UK 27203501).

12. Klenow enzyme, 2 U/|L (Boehringer-Mannheim)

13. Amplitaq (5 U/|L) and 10X buffer: 100 mM Tris-HCl (pH 8.3), 500 mM KCl (Applied Biosystems N8080161).

14. 25 mM Magnesium chloride.

15. Restriction endonucleases XbaI and Kpnl for cloning PCR products.

16. Glycogen, 5 mg/mL (Boehringer-Mannheim).

17. 10 M Ammonium acetate.

18. Absolute ethanol.

20. pBluescript SKII+ plasmid DNA (Stratagene, Cambridge, UK).

21. T4 DNA ligase (1 U/|L) and 10X ligation buffer (Boehringer-Mannheim).

22. Electrocompetent bacteria (e.g., DH5a).

23. LB agar: 1% Bacto tryptone (w/v) (Difco, Detroit, MI), 0.5% Bacto-yeast extract (w/v), 1% (w/v) sodium chloride (w/v), and 50 mg/mL ampicillin.

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