strange sequencing / cloning problem with AvrII-site - (Jan/24/2007 )

Hello all

I experience a very strange issue at the moment... maybe someone can help!

Facts: I TA-ligated a gene (3kb) containing a single AvrII restriction site (about in the middle) into pGEM-T. So far so fine... Sequencing with M13-fwd / M13-rev and a primer that covers the AvrII site is all normal, eg. the RE-site is there. When I ligate a 444bp sequence into this construct (AvrII / dephos / T4 / ON) I get nice colonies and when I screen them i get positive clones (eg. insert in the right direction, one primer fwd outside / one rev inside the 444bp fragment). Now, I miniprep a colony, all nice, good amount of plasmid (12ug).

Now the problems start: I cant anymore amplify my gene by pcr! (I have to mention, before the fragment was inserted, this worked well). But: When sequencing with M13-fwd I get a good sequence (containing the gene's fwd primer annealing site) and with M13-rev as well (for the gene rev primer). Now, when I sequence across the region that should contain the fragment (the primer is located about 100bp upstream of the insertion (AvrII-site) locus I get this: My sequence is fine until just after the fragment starts (the AvrII site is there)! Then signal just vanishes (we are using cycle sequencing on an ABI PRISM)

ANY ideas???? Similar experiences???? polymerase inhibiting secondary structure formation???? multiple insertions (although the colony screen pcr band was the right size)??

Thank you, Pascal

edit: when I recut the thing with AvrII, I cut out nicely the 444bp fragment (and the linearized rest)

i have seen similar results on a beckman-coulter ceq. the explanation from their troubleshooting manual is that there is a physical blockage (eg-hairpin) present.

one way around this is to pre-heat treat your plasmid (1-3 minutes at 96C or 5 min at 86C) in water, before adding your sequencing reagents.

another way around this is to pcr your plasmid for the insert and sequence the pcr product (you can use the same primers).

If you know the sequence of your insert, I would try to sequence from the insert out into the plasmid backbone and see what happens. You could also try adding Betaine or DMSO to the sequencing reaction.