I need help sequencing a plasmid - pretty please, with sugar on top! (Nov/02/2007 )

We were given pKH3 plasmid (4.8 kb) from neighboring lab. We have our own pKH3, but we only have EcoRI-BamHI cut -- I needed a full plasmid to use a different RE. Our same primers work with the "new" pKH3, and there are some minor differences between the two when we sequence. However, these differences are small, and we wouldn't expect any problems from them.

After RE (w/ Eco-RI only, then CIAP or SAP phosphatase treat, gel purify), ligating, and transforming (and getting colonies that have the right size vector and insert bands), it no longer sequences with those primers (forward or reverse... if I send in a primer for the insert, that sequences ok). The very similar "old" pKH3 works fine for clonining/sequencing. I just have a hard time believing that for some reason this RE would ruin the binding site for the primers.

Any thoughts?

Thank you!

where are your vector primers supposed to bind?

In my opinion you've answered your own question, weir as it might be, but if insert primers sequence fine, but vector don't the only explanation (with the information you've given) i can think of is that those primers are binding the site you've disrupted. when using insert primers, do you get enough vector sequence to asses this?

Thank you for the response.

My forward sequencing primer should be binding 193 bp upstream from EcoRI, and my reverse primer should be 64 bp downstream of EcoRI. The primer from the insert doesn't sequence far enough to see any vector DNA.

Thank you again.