DNA length and annealing kinetics I have a mixture plasmids and undesired short linear fragments that share the same sequences. During denaturation and annealing, I would like the plasmids to 'find each other' before annealing to the shorter linear fragments. Assuming the concentration of shorter fragments is significant, is there a temperature profile to bias towards re-annealing of longer DNA? More specifically, this is for a variation of the Surveyor Mutation detection assay, where re-annealed DNA with mismatches are digested, leaving non-mutant DNA intact. I would like to keep non-mutant plasmids for E. coli transformation. However, some linear fragments ~10-50% of the length of the plasmid have escaped exonuclease treatment and would compete with the plasmids during annealing.

Short answer is that higher temperature favors annealing of longer sequences. There are number of ways to calculate melting temperature, but all of them produce similar results: longer polymers require more thermal energy to melt. Hence, **quick** cooling from higher (say, from 95C thermocycler can cool in 10-12 sec) to RT/4C will favor re-annealing of circular strands. Slower cooling should allow more ssDNAs to bind.

But again, if end goal is to select for circular dsDNA, simple transformation should take care of that.