In general, increased reaction time, lowered reaction temperature, and molecular crowding all yield more complete ligation reactions. Reaction times can be increased as long as 24 hours or more. For ligations longer than 2 hours, we routinely incubate below 16°C. Some scientists report ligation on ice (0°C) for 24 hours. We recommend the use of PEG 4000 or 8000 up to 15% (w/v). Increasing PEG concentrations further did not further increase ligation efficiency.
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