Dark Banding in Southern Blot

[BLACKBEAKER]

Hi,

Recently I have been some work on some putative double cross-over mutant constructs which have demonstrated the correct antibiotic profile after transformation and selection (phenotype unknown).

However, when we PCR these strains it appears that the gene is not interrupted and the plasmid backbone is still present. To resolve this (i.e - if it was a single crossover insertion instead of a DCO) we decided to undertake Southern blot analysis digesting with HindIII to generate products of a known size.

Yet when we do this we observe in our DNA digests two very strong bands (see attached pic) of weights that do not match to what we predict for SCO/DCO or replicating plasmid. And when probed the bands are shown to contain our gene of interest.

The bands only appear in the mutant strains and not the wild-type control; which through both southern and other means we have shown are not replicating plasmid (as would be expected by such high copy number of these bands), suggesting they are integrated in some way.

We are completely lost as to what may be the cause of this and I would be interested to here if anyone has had a similiar experience or know's what may be the cause.

Thanks heaps

Beaker