I have reason to believe (details see below) that in a ligation I carried out, an EcoRI sticky end (EcoRI: G'AATT_C) and an XmaI sticky end (XmaI: C'CCGG_G) were somehow ligated together, a process during which at least the EcoRI site was lost: the plasmid had a BamHI site very near the XmaI site, and a BamHI/EcoRI digest produced only one band compared to three bands in the undigested control (three conformations of circular plasmid).
How could ends as incompatible as EcoRI and XmaI be ligated?
Detailed Background or: Why I believe incompatible sites were ligated
This is chronologically further to: What are common causes of unexpected ligation products?. Briefly: I digested two plasmids, one with EcoRI and XmaI (p1), the other with EcoRI and AgeI (p2) [XmaI and AgeI produce compatible sticky ends], then carried out a ligation between a 1.4kb insert isolated from p2 (structure EcoRI-1.4kb-AgeI) into the 3.4kb backbone isolated from p1 (structure XmaI-20bp-BamHI-3.4kb-EcoRI). After transformation, I tested the ligation product by digest with EcoRI and BamHI, which should produce again 3.4kb + 1.4kb. The gel was poor quality but allowed the conclusion that there were different ligation products.
Repeating the EcoRI/BamHI digest and gel more carefully showed there were only two variants: Variant A (8 colonies) produced the two expected bands 3.4kb and 1.4kb. Variant B (4 colonies) only produced a clear 3.4kb band, nothing else visible in the lane: in other words, it only carried either an EcoRI or a BamHI site and was in total smaller than variant A (confirmed by undigested controls). Thus, I conclude that B was simply the same 3.4kb backbone, re-ligated without the 1.4kb insert. Since the BamHI site was untouched in the backbone during the preparatory EcoRI/XmaI digestion, I assume that the EcoRI site was not recovered during the ligation of variant B.
(The ligation was carried out overnight at 16 deg C, using Promega T4 DNA ligase and buffer and a molar ratio of 3:1 insert:vector at a total volume of 20uL. The E.coli for transformation were Invitrogen OneShot Stbl3 and have been routinely and successfully used in the lab for a long time. The analytical digestion used Promega EcoRI and BamHI in Buffer Multi-Core, which Promega claims offers 75-100% efficiency for both enzymes. Incubation was 1.5h at 37°C.)