1. The reaction mix is assembled as follows:
xx pL first-strand reaction (~125-150 pL);
30 pL 10X second-strand buffer;
30 pL 20 mM dNTPs;
xxx pL H2O—to a final volume of 300 pL;
2. Incubate overnight at 14°C.
3. Heat at 80°C for 15 min, cool on ice, and spin briefly to collect the liquid.
4. Add ~20 U of T4 DNA polymerase, and incubate 1 h at 37°C (see Note 2).
5. Phenol extract, ethanol precipitate, rinse, and dry.
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