DNA linear transformation failure - (Nov/21/2006 )

We are trying to do a DNA linear transformation using a 5.5kb PCR product (gene promoter+lacZ+cat gene+ 40bp of putA homology sites) in Salmonella typhimurium. The problem is we are getting no transformants. We tried adjusting the volume of arabinose and DNA during the process, but still it failed. Anothet student spent 4 months trying to get a transformant but didnt succeed. Now, the task is mine. I tried repeating all the experiments and already spent 2 weeks for it but still no success. I dont know where the mistake is.

Our goal is to study the gene regulation of that particular gene. Anyone who has experience using the same technique pls enlighten me. My motivation is getting low.

Thanks

We are trying to do a DNA linear transformation using a 5.5kb PCR product (gene promoter+lacZ+cat gene+ 40bp of putA homology sites) in Salmonella typhimurium. The problem is we are getting no transformants. We tried adjusting the volume of arabinose and DNA during the process, but still it failed. Anothet student spent 4 months trying to get a transformant but didnt succeed. Now, the task is mine. I tried repeating all the experiments and already spent 2 weeks for it but still no success. I dont know where the mistake is.

Our goal is to study the gene regulation of that particular gene. Anyone who has experience using the same technique pls enlighten me. My motivation is getting low.

Thanks

Thanks for the reply. Ill look into that