Insert crashing ligation?

[elsancheezmo]

Hi - I'm setting up (3) ligations. Vector only, + 1x insert, + 2x insert. I get colonies on my background transformation but nothing on my insert plates. I would expect at least the same number of colonies on all plates even if the cloning failed since the vector input is the same......HELP!

[keithbrent2000]

Maybe your vector is ligating back onto itself, especially in the absence of an insert, which is causing the colonies to be present. Perhaps you could try 3x insert and 1x vector. Here are also some more [Only registered users see links. ].

[Aggie]

I am having a similar problem and discovered that my vector is not being completely digested. I can only detect it when I transform I cut and phosphatased vector sample that was not treated with ligase. I get as many colonies on those plates as my ligase treated samples. I have increased the units of restriction enzyme (SphI) and the length of incubation with no improvement. Any suggestions?

I have used as much as a 1:10 (vector:insert) ratio successfully for my ligations. I would no try lower than 1:3. You can also treat your vector with a phosphatase to prevent self-ligation.