Hi Jonathan

It sounds as if you are trying to do a gene knockout or allele replacement using linear transformation. The fate of electroporated linearised DNA is commented on [here]. It is of couse possible to electroporate linear DNA, though it is quite an inefficient process.

What you're usually looking to do is simply get enough molecules into the cells (usually quite a high concentration) so that in the event of effectively having a cells that is merozygotic for a particular gene, an recombiantion event will hopefully lead to the replacement or insertion of the transformed gene into the chromosome (in this context sometimes referred to as the endogenate). Until this point, the linear DNA is subject to exonuclease host-restriction mechanisms that are designed specifically to degrade exogenous DNA.

The transformation of E. coli with linearised plasmid DNA can result in the formation of viable plasmids as long as a) there aren't any host plasmids with which the introduced origin of replication (oriR) is incompatible, and b) you don't mind ending up with something that may not look like what you expect. Linear DNA is uprotected, both in terms of DNA binding proteins and the free nucleotide ends. The fragment is subject to host enzymes (depending on the strain) that may reuslt is a chimeric DNA molecule that isn't necessary what you expect.

If you were to be attempting gene knockouts or replacement, then ideally you would be transforming a PCR product of a suitably engineered gene alternative. This would be highly purified and the final PCR performed using the previous PCR fragment as a template. An example is if you were trying to replace a host resistance cassette with a diffent one. When you select for the introduced resistance, you want that to be due to the successful introduction of your cassette into the host genome, NOT because you have a plasmid containing the resistance cassette and a viable oriR.

As this is potentially quite a technical question, and depends very much on a) the experiment you are trying to perform, and b) on the strain of E. coli with which you are working, my best help would be to refer you to a [molecular biology forum]. Here you can receive helpful practical advice with a rapid turn round, as an alternative to the largely academic nature of the Madsci site.

Hope this helps

Jim
MAD Scientist.