AlwI NEB#R0513

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	The PCR products were separately digested with the corresponding restriction enzyme (MboII for 3′-half and AlwI for 5′-half) and then ssDNAs were collected through avidin-biotin binding (streptavidin-coated magnetic beads were used)

Protocol tips
The PCR products were separately digested with the corresponding restriction enzyme (MboII for 3′-half and AlwI for 5′-half) and then ssDNAs were collected through avidin-biotin binding (streptavidin-coated magnetic beads were used)

Publication protocol

The pre-5′-half and the pre-3′-half PCR products were prepared using the ligation products recovered by ethanol precipitation. Each DNA was incubated with 10 U restriction enzyme [MboII (Takara), AlwI (New England Biolabs, Beverly, USA) or MboI (Takara)] in 10 μl of a buffer {MboII buffer [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT], AlwI buffer [20 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT and 50 mM KCl] and MboI buffer [20 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT and 100 mM KCl]} at 37°C for 1 h. The surrounding sequences of the recognition sequences are shown in Figure 1

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