Table 1.

Optimization of random mutagenesis of horse heart myoglobin by PCR

PCR protocol, MnSO4	Nucleotide substitutions	Amino acid substitutions
0.50 mM/35 cycles	6–10	4–6
0.25 mM/25 cycles	4–6	2–4
0.13 mM/25 cycles	2–4	1–2
0.06 mM/25 cycles	2	1

The PCR protocols for random mutagenesis were modified on the basis of routine PCR [a 100 μl reaction mixture contained 67 mM Tris⋅HCl (pH 8.8), 16.6 mM (NH₄)₂SO₄, 6.7 mM MgSO₄, 0.07% β-mercaptoethanol (vol/vol), 200 μM each of dNTP, 0.1 μM each of primers, 10 ng template DNA, and 2.5 units Taq DNA polymerase] by adding varying amounts of MnSO₄ and running different number of cycles.