E.coli + pCAMBIA1302 - everything is more complicated than expected... (May/30/2007 )

Hi everybody!

My firat attempt to clone pCAMBIA1302 in E.coli was a disaster. Can you please give me your advices?

I've eluted pCAMBIA1302 from the paper blot and checked the elution. I amplified a fragment of the mgfp5 gene then made an electrophoresis run. Everything was ok.

I followed Promega instructions performing E. coli JM109 transformation by heat shock. Then I plated the bacteria suspension on LB agar plates + 50 mg/L kanamycin (as pCAMBIA includes aadA kanamycin resistance gene to screen bacteria) and let them incubate over night.
With my great disappointment - on the next day I couldn't find a single colony.

In the same time, I was performing a second E. coli transformation to insert an amplicon in JM109 plasmid. That cloning was successfull, so I guess the bacteria stock is fine.

Can my problem depend on the antibiotic formulation? Is something wrong with the procedure?

I am quite lost and I will treasure any advice!!!
Thanks a lot!

ILA

Are you growing your transformed bacteria up without kanamycin for an hour prior to plating them out? Kan plasmids require time to express the resistance gene.

I really didn't know that!!!
Could you please explain me the molecular reason?

Thanks phage!

Resistance to antibiotics requires the production of a specific protein in the cell. The plasmid contains the gene for this protein. After the plasmid is inserted, the protein requires time to express. For some antibiotics, such as ampicillin, the antibiotic is not lethal during this period, but rather inhibitory for growth. For others, such as Kan, and especially Tet and chloramphenicol, the protein has to be there from the start. For these cells, we transform and then grow the cells up in non selective, rich medium (typically SOC) for 1-2 hours, allowing time for protein expresson, then plate them out on antibiotic selective plates.

Thanks for the exaplanation...I didn't think about this aspect...

Anyway, I let bacteria to grow in SOC without kanamycin for almost 2h .
So, this is not the case ...

...other tries???