CDNA Synthesis

1. 10X First-strand buffer: 0.5 M Tris-HCl, pH 8.9 (this will be 8.3 at 42°C), 0.1 M MgCl2,0.2 M KCl, 50 mM dithiothreitol (DTT).

2. 10X Second-strand buffer: 0.2 M Tris-HCl, pH 7.5, 0.05 M MgCl2, 0.1 M (NH4)2S04,1.0 M KCl.

3. 10X EcoRImethylase buffer: 1.0 M Tris-HCl, pH 8.0, 1.0 M NaCl, 0.01 M EDTA. Make as a stock solution and then add S-adenosyl methionine to 0.8 mM to a small aliquot of this stock before use.

4. 10X T4 polynucleotide kinase buffer: 0.5 M Tris-HCl, pH 8.0, 0.1 M MgCl2,50 mM DTT.

5. 10X T4 ligase buffer: 0.3 M Tris-HCl, pH 7.4, 0.04 M MgCl2,0.1 M DTT, 2 mM ATP. Make ~10 mL of this buffer and store in 100-pL aliquots at -20°C. Repeated freeze-thaw cycles rapidly deplete the buffer of both DTT and ATP.

6. dNTP mix with 5-methyl-dCTP: 20 mM dATP, 20 mM 5-methyl-dCTP, 20 mM dGTP, 20 mM dTTP.

7. dNTP mix: 20 mM dATP, 20 mM dCTP, 20 mM dGTP, 20 mM dTTP.

8. 10X EcoRI buffer—supplied by the manufacturer: 0.5 M Tris-HCl, pH 7.5, 0.1 M MgCl2,1.0 M NaCl, 10 mM DTT.

9. Column buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.15 M NaCl.

10. TBE for electrophoresis (10X): 0.89 MTris base (Boehringer Mannheim [BMB], Indianapolis, IN), 0.89 M boric acid, 0.02 M EDTA. Filter and autoclave (prevents precipitate from forming with time).

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