Hi I've digested two vectors with XhoI. Vector one has two XhoI sites and its digestion allow me to recover the insert that I should clone within vector 2, which instead includes only one XhoI site. In order to avoid recircularization of vector 2, before loading both restriction reactions on agorose for cutting the corrected bands,I treated it shrimp alkaline phosphatase (SAP). Then I load everything on agarose and cutted out the two bands (about 700 bp and 5 Kb). After that I ligated the two fragments using different molar ratios (10:1, 3:1, 1:1, 1:3 Insert:Vector) and transformed Ag1 E-Coli strain. As lower the amount of insert I used as much transformed colonies I obtained, but in all cases the colonies did non include my fragment. I loaded the ligation product on an agarose gel and what I saw were multimer of insert (that demonstrates that both ends of this fragmet are able to ligate to each other) but no evidence of circular plasmids. I don't know how can I fix this problem. Do you think I should change the molar ratios insert:vector or should I think that SAP may alter the ends of my vector?