反应详情

The subtracter-amplicon was diluted to 10 μg/ml, and four PCR reactions were set up on ice to generate subtracter U-DNA for the control set. Each 50 μl reaction contained 2 μl diluted ligation, 1 μl 4 μg/μl S-hpa-24mer oligo, 2 μl dNTPs (10 mM dATP, 10 mM dCTP, 10 mM dGTP, and 30 mM dUTP), 5 μl 10× PCR buffer, 1 μl 3.5 U/μl Taq DNA polymerase and ddH2O. The S-hpa-12mer was melted away (5 min, 72° C.), and ends filled in with Taq DNA polymerase (7 min, 72° C.). Twenty-one cycles of amplification were performed (1 min, 95° C.; 3 min, 72° C.), and the products were phenol-extracted, isopropanol-precipitated and resuspended in 20 μl 10 mM Tris-buffer each (Sambrook et. al., "Molecular Cloning, 2nd Edition", p10.49 (1989)). The S-adaptors were then removed with Hpa2-restriction, and the restricted products (subtracters) were phenol-extracted, isopropanol-precipitated, combined and resuspended in a total 16 μl EEx3 buffer (30 mM EPPS, pH 8.0 at 20° C.; 3mM EDTA). 6 μl (9 μg) subtracters were measured on a 2.5% TBE-agarose gel.

1. additionThe subtracter-amplicon was diluted to 10 μg/ml, and four PCR reactions
2. customEach 50 μl reaction
3. custom(5 min, 72° C.)
4. custom(1 min, 95° C.; 3 min, 72° C.)
5. extractionextracted
6. customisopropanol-precipitated
7. customThe S-adaptors were then removed with Hpa2-restriction
8. extractionextracted
9. customisopropanol-precipitated
10. customresuspended in a total 16 μl EEx3 buffer (30 mM EPPS, pH 8.0 at 20° C.; 3mM EDTA)