Working at the wrong temperature. β-Agarase I is quickly inactivated at temperatures above 45°C. The low melting point agarose must be melted at 65°C then equilibrated at 42°C. Therefore, when working with large volumes, be sure to leave ample time for molten agar to equilibrate to 42°C. At lower temperatures the agarose starts to solidify making it resistant to digestion.