Alright, so I'm constructing a deletion mutant, basically I've amplified upstream + downstream regions, and put a gentamicin resistance cassette in the middle and transfered this on a plasmid to my bacteria wild type strain for homologous recombination.
I got what I thought were mutants and preped genomic DNA from them to confirm the mutation. I used primers to amplify upstream + downstream regions of where the deletion should be. The primers I used are located even further upstream + downstream of the upstream and downstream regions amplified for the recombination event (ie the upstream + downstream regions with the gentamicin resistance cassette in the middle).
The predicted bp size should be 3.4 bp, however it's something like 4000 bp. I have no idea what could possibly be happening and where these extra bp are coming from. I really have no idea what to do. Any suggestions as to what might be going on?