I am currently performing ligation of my Bcl1 digested product and for one reaction I have to add 5U of T4 DNA Ligase per 250uL of total reaction volume. The enzyme I recieved, had explained its units as 200CEU/uL or 1 Weiss unit/uL. Anyone can guide me the conversion of Weiss unit to unit? To achieve 5U of the enzyme how many uL I should consider adding?
There is not a standard definition of a Unit for Ligase, so when you say 5U that doesn't really mean anything without defining the unit. Fortunately it doesn't hurt to have a bit of excess ligase, so I would add about 1 ul to your 250ul reaction and you will be in excess.
This is a large volume with 250ul for the total reaction. When I clone, I use up to 20ul of total reaction and use 1ul of T4 Ligase.