A1: AflII is inhibited by salt concentrations greater then 50 mM. Some mini prep DNA may contain residual salt. A 70% ethanol wash or dialysis is recommended to remove the salt.
Q2: Why don't AflII- generated DNA ends ligate well?
A2: We don't know. There is something odd about the DNA conformation of these ends.
Q3: How can ligation efficiency be increased?
A3: Less than 50% of AflII fragments ligate in a standard 20 microliter reaction containing 100-500 units of T4 DNA ligase. Ligation can be enhanced by using 1 microliter of high concentration T4 DNA ligase. Blunting the ends, wither by fill-in or removal, increases ligation of the poorly- ligated ends.
Q4: What is the molecular weight of AflII?
A4: The MW of AflII is 36,778 Da from sequence data.
