I tried to ligate an insert (1.5 kb) with an unphosphorilated vector (4.3kb) with 2 different sticky-end enzymes (KasI and XbaI).
Ligation yielded much more colonies in the positive (vector+insert) than in negative plate (only vector)
Colony PCR was positive for all 25 clones. Forward primer is in vector and reverse in insert.
Restriction digestion with the same enzymes (KasI and XbaI) showed only a single band with the expected size of the vector but no insert at all.
Has anyone any idea of what could be happening?
Edited by tapi, 10 October 2013 - 07:06 AM.