Normally people trust the replication machinery of E. coli and don't resequence their plasmids, unless there is a specific reason for it : unstability due to direct repeats, mutagenic host, expression of a toxic protein etc. But if the transformed strain behaves in a totally unexpected way, resequencing will be advisable: extraordinary results require extraordinary strong proofs

I agree with Pierre Béguin . Usually if it is a simple transformation people tend to trust that it is correct after the initial confirmation. However, in certain cases (unstable or toxic sequences) causes rearrangements in the plasmid. An obvious sign of toxicity is getting colonies of various sizes after the transformation. I suggest you confirm your plasmid with a restriction digest in addition to sequencing the insert to confirm that there are no gross rearragments of the non-insert part. I would recommend that you freeze your plasmid both as a glycerol stock (for rapid extraction) and as DNA so you can go back and retransform in case you run into any problems.

If you have PCR primers for your cloned DNA fragment (in pricipal you should have) than can you easily control your strains by direct colony PCR. Take a single colony from the edge of a culture plate and perform the control PCR. You can also use the sequencing primers designed for the cloning site. But I think that it makes only sense in the early phase of your cloning project. Later the strains (growing on the appropriate selection medium) should be ok. Otherwise I agree with Pierre. Make glycerin stocks and freeze them in -80. Then you store them 100 years :-)

following

If you're concerned you can do a restriction digestion of purified plasmid from your newly transformed cells.

Or, if you're confident in your plasmid construct you can simply do a colony PCR using one primer from the plasmid vector backbone and one from your insert. A lot of people will have generic plasmid vector primers (T7 and M13 depending on the plasmid vector) laying around in a freezer so I'd say send round an email if you don't have any. You can use the cloning primer for the insert. Colony PCR is a quick and dirty method so I'd use appropriate positive and negative PCR controls e.g. the construct of interest, the insert, an empty plasmid vector and a no template control.

That would give you all the data you need to be very confident your construct has transfected and the PCR amplified your target.

This technique does assume your primers have the same annealing/melting properties. It's a good idea to try and standardise your primers to a general Tm. This can be difficult if you're aiming to overexpress a gene.

Also, do an in silico PCR first to make sure you're using the correct PCR primers i.e. you're not using two positive strand primers.

After confirmation by sequencing, you should make glycerol stocks and freeze them in -80. Then whenever you will be in need of using that plasmid, you just have to culture from glycerol stock. To be sure you can check it by restriction digestion.

Agree with the earlier comments, Pierre Béguin, Hanna Alalam, Frank Dunemann.

Since you have transformed your desired plasmid (with selective marker) into the E. coli host and confirmed with the sequencing, I think it is not required to confirm by sequencing every time unless an until host is behaving differently. E. coli is considered as a model host and most of the vectors are not lethal to the host. Obviously if the clones are growing on the selective plates containing markers (e.g. Amp/Hyg etc.) that means hosts are carrying your desired plasmid.

But yes, you can confirm your inserted gene of insert by targeted sequencing using vector specific sequencing primers after every 3-4 generations. Here, no need to sequence vector backbone again.

Yes, i agree all that you can trust the appropriate selection for your transformants and if having doubt then you can used any of the suggested method. Sequencing should be the last choice.