egfp in ecoli (pet60) - (Dec/11/2008 )
I'm just an engineer, so I'm having a little bit of trouble with the basics.
I got some ecoli from a friendly lab nearby (DH5alpha) and some egfp plasmid (pet60)? so that I can prep enough plasmid to transfect some cancer cells but I'm having some trouble finding out the details on this plasmid, and the lab that they came from hasn't been able to explain them to me.
it does have amp resistance because my colonies are growing on an amp plates but when i check them under the fluorescent microscope they dont look like they are expressing gfp?
is it common for a plasmid to have a promoter/ribosomal binding site / (other fancy things that I dont understand) that would be specific to mammalian cells and thus would not be expressed in ecoli?
I just want to be sure that the colonies growing on the amp plate actually do have the plasmid with the egfp gene.
I haven't worked with pet60 in particular. But mammalian proteins generally will not be expressed in bacteria. That's why there is an antibiotic resistance gene in the plasmid, in your case ampicillin. If the e.coli are growing on the amp plates, they should have the plasmid (assuming your amp plates aren't too old).
Qiagen has some useful general info about plasmids on their website:
Hope that helps!