I have an insert with RE sites including 6 bp flank from the ends. The RE sites are MluI and BssHII, in a way that both sites are compatible with MluI digested Vector.

After digesting the vector, i confirmed its appearance of a single band at 3Kb as opposed to 3 bands of different forms (Of circular DNA), when intact. Vector was then Treated with CIP and purified with Phe:Chcl3. LIgation of Purified MluI and BssHII digested with Phosphate treated Vector Yielded only wild type colonies (of the self ligated vector). The colonies were:

1. Vector control (Vector put in Ligation Rxn without Insert) : 12 colonies/ul of cultured (for 1h) transformed cells (DH5alpha/NEB comp cells).

2. V+I (Vector put in Ligation Reaction with Insert) : 16 colonies/ul.

Plating was done with antibiotic that is present in the vector backbone (ampicillin).

Colony PCR was done with Primers that flank the insert site, so we would have recombinants with larger bands.

from this, i thought that Phosphatase treatment was not sufficient. So, i treated the CIP Treated MluI cut vector with Shrimp Alkaline Phosphatase (rSAP). Ligation was done using the same procedure with Quick Ligation kit and transformation in NEB DH5Alpha comp cells. For transformation 2 ul of Vector control Ligation reaction (in which Insert was not added) and 2 ul of Vector+Insert Rxn was used for transformation. The colonies i got now are:

1. Vector control (Vector put in Ligation Rxn without Insert) : 0.8 colonies/ul (39colonies/50ul) of cultured (for 1h) transformed cells (DH5alpha/NEB comp cells).

2. V+I (Vector put in Ligation Reaction with Insert) : 14 colonies/ul (720colonies/50ul).

Looks good. right? that's what we thought.

But with colony PCR, all the colonies in V+I Reaction having 720 colonies on plate, were found to be wild type colonies! i took 48 colonies from those 720 to screen recombinants. Please Help What is there that i am doing wrong.