I am digesting a 12kb plasmid that I have constructed with NdeI. I THOUGHT the plasmid had 3 NdeI sites and as a result would yield 3 bands totaling 12kb. I will be sequencing the plasmid to make sure I have the correct sequence. But what I observed is two bands totaling 12kb. So, it seems I have two NdeI sites and the third site has mutations. My question is:
Is it possible for there to be 3 NdeI sites, but only have 2 of them cut efficiently? In this scenario all of the enzyme would bind the 2 "efficient" sites and the 3rd inefficient site would be left uncut. I am just trying to see if anyone has observed this. I am sequencing the 12kb plasmid to find out definitively, but I really hope there are not mutations, as it took a lot of work to construct this, and it feels like a slap to the face when I have to correct mutations.
The digest conditions were
2uL 10X buffer
H2O to 20uL