pUC4K HincII sites, how many? - (Aug/28/2011 )

btw, to be exact and complicate things more, the bands are @:

1000bp
1250bp
1750bp

to be honest ...never worked with pUC4K

another explanation would be a third HincII site introduced by a point mutation since HincII has a degenerated recognition site ...would be the only logic answer when you are sure your bands add up to the right size of 3.9kB (i'm not really used to your marker ...so you will know best what size the bands are).

Maybe the vector was subcultured to often ...mutations do happen ...i remember a case in our lab where we bought a plasmid from a vendor that starts with Clone and ends with tech ...the nuclotide sequence of the vector said that a certain restriction site was present and we were not able to cut the vector with that enzyme ...in the end we we proofed by sequencing that the original plasmid we bought had a point mutation that leads to the loss of the restriction site. It's biology

Why do you want to remove the Kan resistance?

Regards,
p

I need it to replace a gene ...

no alternative restriction sites?

Check this old article out, it has a map of pUC4K with 3 HincII sites, amazing! but not detailed enough, alas.

BROOME-SMITH, J. K. (1985). "Construction of a mutant of Escherichia coli that has deletions of both the penicillin-binding protein 5 and 6 genes." Journal of general microbiology 131(8): 2115.