Second Strand Synthesis

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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