Plasmid digested with RE used to transform...what will happen? - (Aug/27/2012 )

p-GEM-T with nlgn3 insert in circular form.

Linear DNA gets digested by the endogenous DNases both secreted by, and inside the bacteria. The plasmid, if it transforms should still express B-lactamase, provided the digestion hasn't cut in the gene or its promoter. You can check the location of restriction sites quite easily on the web by simply googleing the name and looking at the sequences that you should be able to find. pGEM-T comes with a product manual that contains a lot of information, including the restriction sites.

@bob1: if linear DNA is always digested by the endogenous DNases, how does one transform the product of ligation independent cloning?

@Andreea: I was under the impression that this form of cloning was more or less equivalent to nicked circular plasmid, rather than linear?

@bob1: yes it is double nicked DNA; mea culpa: I was referring to the fact that when you digest a plasmid with a RE, it's like you have a circular double-nicked plasmid. However, to shoot myself in the leg: in the case of RE you have short sticky ends: 3-6 bp; which are not enough to keep the region annealed at 37oC long enough for the bacteria to repair the nick. My scenario would be feasible if we would have REs that produce ~12 bp sticky ends. Wouldn't life be nice then? People developing ligation independent cloning would not have to suffer by optimizing DNA polymerases to produce the sticky ends

Andreea