Troubles with vector dephosphorylation - troubles with vector dephosphorylation (Feb/17/2006 )

I am trying to insert a ~5kb parts of C.krusei genome into a pBluescriptSK+ vector. The trouble is that after ligation the vector and an insert colonies do not grow at all. Even those with closed vector.I suppose that the problem is with vector. After digesting (BamHI) I am doing a dephosphotylation its ends using Calf Intestine Alkaline Phosphatase ( 1u CIAP for 50 microliter total volume, included 40 microliter of vector DNA, 1h in 37). After ligation, using only the vector DNA as a control of dephosphotylation, and transformation into cells I expected few blue colonies, but I had no colonies on my plates ( LB + amp 90 micrograms/ml). Is the time of dephosphorylation too long or maybe the concentration of CIAP is to high? Thank you for an answer

hi
1h is nnot too long and amount of CIP is not too high.
you may have a pb in logation rather than in dephopshorylation as the control vector do not grow too.
Try to reprepare vector and good purification is better.