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Competency cells analysis - (Dec/10/2011 )

I prepared labmade Agrobacterium (LBA 4404 and AGL1) competent cell and to verify the competency I used a closed cloning vector and I could evaluate it by heat shock. However, when I used a closed expression vector (I will transform rice cells using this vector harboring my gene) I got nothing, no cells at all. I do not know why this is happens and what I have to do to analyse the competency of the cells and use it in my transformation experiments. Would someone please help me on that? Thanks! Angela

-Angela Vitoria-

Have you checked the preparation of the expression vector by gel as well as determined the concentration? You should do this to ensure that you are not transforming large quantities of genomic DNA which could be present in your plasmid prep.

Have you tried transforming the backbone of this expression vector (i.e. just the plasmid without any genes added)? If you are just growing up plasmid for later use, why not use a high efficiency E. coli competent cell like DH5alpha?