I selected a single restriction enzyme cutting site BamHI in a plasmid of around 6 kbp, performed enzyme digestion, and carried out dephosphorylation treatment. I attempted to insert a fragment of approximately 1.7 kbp, for which a BamHI restriction site had been added through PCR and subsequent enzyme digestion. After ligating the two fragments and transforming them into Escherichia coli, colonies formed following antibiotic selection. However, upon further verification through colony PCR and enzyme digestion, it was observed that the 1.7 kbp fragment was not present in the plasmid. Despite multiple attempts, this result persisted. I would like to ask if anyone has any suggestions or improvements for the procedure of inserting fragments into single restriction enzyme cutting sites. Your insights would be greatly appreciated.
What happens with your control ligation (vector but no insert and insert but no vector)? How many colonies do you get with those compared to the actual ligation product.
Did you add a few spacer nuts on either side of the BamHI site in the primers to be sure that digestions goes efficiently?