A2: Because there is an exonnuclease or nonspecific endonuclease acitivy that appears to be a property of the enzyme.
Q3: Why is AlwI- cut DNA difficult to ligate?
A3: Single base-pair overhangs are poor substrates for T4 DNA ligase: they anneal very poorly, and are as difficult to ligate as are blunt ended DNA fragments. Also, for AlwI the overhang is not constrained to be any particular nucleotide; all four bases are roughly equally represented. Consequently, any given overhang is compatible with only 25% of the other overhangs present. This lowers ligation efficieny relative to overhangs generated by restriction enzymes which leave only mutually compatible overhangs.
Q4: How can I increase ligation efficiency?
A4: Use high concentration DNA ligase.
Q5: What is the activity of AlwI at 25°C?
A5: AlwI is 25-50% active at 25°C.
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