EcoRI Methylase Treatment

1. Resuspend the cDNA in 85 pL of H2O. The reaction mixture is as follows:

85 pL cDNA;

11 pL 10X methylase buffer;

10 pL EcoRI methylase (100-400 U). Incubate for 30 min at 37°C. Add 4 pL more methylase and incubate for 30 min longer.

3. Add 1 pL of 20 mM dNTP's and 1 U of T4 DNA polymerase. Incubate for 30 min at 37°C (see Note 3).

4. Phenol-extract, ethanol-precipitate, rinse, and dry the cDNA.

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