I am trying to use double selection marker, G418/kanR and BleoR in pPICZalpha plasmid. I am constructing the plasmid with both antibiotic resistance gene and clone it into E. coli Top10. However, I cannot get any colonies in LB low salt KanR BleoR plate. I only know that LB low salt plate is required for BleoR in E. coli. Is there anything else wrong? Any suggestion is welcome and THanks!
Hi
Have you inserted the promoter of two antibiotics upstream of the antibiotic gene?
I would suggest you make some plates with each antibiotic and see if your clones work and confer resistant on the individual antibiotics first (Bleo being the more likely problem). That way you can at least identify the problem. If the individual antibiotics both work but the double selection not, you may just be at the limits of resistance and cutting back a bit on the concentrations in the dual selection plate might help.
In continuation of Dr. Michael J. Benedik's suggestion, based on a similar experience I had, I recommend that if you do not have a clone, increase the incubation time (for example, instead of 18 hours t, 24 hours).
some times for some cases, it works.
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