Plasmid problem - (Sep/20/2001 )

Please help me. I am trying to clone genes by PCR, put them into plasmid vectors, do preps on the cells, linearize the plasmids and make RNA probes for whole mount in situs. Only about half of the genes I have cloned work as far as making RNA probes. My preps and linearizations end up looking really weird when I run them on gels. Tech services of several companies have told me it could be that my insert is toxic to the cells or a rearrangement is going on. Has anyone had a problem similar to this? If so, please tell me you have a solution. (best idea I have so far is to just try different strains of cells i.e. SURE cells from Stratagene) HELP! We have been pulling our hair out over this problem. Thanks in advance for any help you can provide.

Although you did not mention the name of the plasmid that you are using let me make a guess :is it pcDNA3 that you are doing the cloning in?We are also having a tough time on similar lines.sankar

I had a similar problem. I used stratagene cells and they gave difficulties growing. I used Gibco electromax cells and they gave good results.