Characterizing an unknown plasmid - (Oct/03/2012 )
I'm in a terrible soup. Received a plasmid construct nobody has a clue about except for its name. It is a GFP tagged plasmid , vector backbone and selection antibiotic , sequence information, and organism from which the sequence was obtained are all unknown or unavailable. How can I identify what backbone the plasmid may be in ? Is it possible to identify sequence using generic primer against GFP ? In urgent need of help !!
A primer aganst GFP should work - put one at each end of the GFP and sequence out. Backbone identification might be more difficult as a lot are not in any sequence databases that I am familiar with. You could screen for resistance by making a plates with common antibiotics and screening the plasmid.
Surely the person who supplied it can provide some information?
Most plasmid backbones have M13 primer sites (forward and reverse) surrounding the cloning site. If I truly knew nothing, I would transform and plate on a variety of antibiotic plates (amp, tet, kan, chloramphenicol probably). Amp and Kan are probably the most likely. I would prep DNA and try sequencing with M13 fwd and M13 rev and primer walk.
thanks !! I'll try all that
what are the usual concentrations of chloramphenicol and tetracycline used for selection (like 100 microg./mL ampicillin or 50 microgr/mL kanamycin) ?
35 ug/ml for chloramphenicol, 15 ug/ml for tetracycline.
You received already a lot of good advice here, and I just want to mention one option: If you use the plasmid to express a gene and if you know what gene it is, you could try to amplify the gene with PCR for cloning into a standard plasmid in your lab. This would not only allow for easy sequencing after the cloning but over time you would acquire all your genes in the same standard vectors which makes your life easier...