Constructing deletion mutant, problems confirming - (Dec/12/2013 )

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?

Hi,

What is the size if you amplify with your gene to be deleted. What is the gentamycin size? Did you calculate the size of from the upstream primer till the downstream primer (because you might have designed the confirmation gene from the upstream and downstream genes). Or any possibility of your primers to sit in different place?

The obvous next step is to sequence your pcr product from each end.