4 replies to this topic

[cell_man]

Hi,

I have a vector containing kanamycin resistant gene which I am trying to transform in to E.coli.  Can I grow them in agar plates coated with Geneticin?

Thanks in advance.

[lab rat]

Hi cell man,

I was taught that kanamycin and geneticin are not interchangeable. Kan is specific for prokaryotes, and geneticin is for eukaryotes, even though both antibiotics act in a similar way. If your plasmid encodes resistance for both, your bacteria will express kanr and not genr. The mammalian cells transfected with the same plasmid will express genr.

regards,

lab rat

[cell_man]

lab rat, on Dec 14 2009, 07:23 PM, said:

Hi cell man,

I was taught that kanamycin and geneticin are not interchangeable. Kan is specific for prokaryotes, and geneticin is for eukaryotes, even though both antibiotics act in a similar way. If your plasmid encodes resistance for both, your bacteria will express kanr and not genr. The mammalian cells transfected with the same plasmid will express genr.

regards,

lab rat

Thanks for the reply. Surprisingly enough I got a colony on the geneticin coated plate. i cultured it in the genticin LB broth and it grew successfully. i could even do miniprep, but my luciferase in the plasmid could not be expressed after transfection even though positive control worked. is that colony not trustable? is something wrong with the colony? why did it grew on geneticin coated plates? the plasmid contains kanamycin/neomycin genes.

TIA

[bob1]

cell_man, on Dec 22 2009, 03:36 PM, said:

Thanks for the reply. Surprisingly enough I got a colony on the geneticin coated plate. i cultured it in the genticin LB broth and it grew successfully. i could even do miniprep, but my luciferase in the plasmid could not be expressed after transfection even though positive control worked. is that colony not trustable? is something wrong with the colony? why did it grew on geneticin coated plates? the plasmid contains kanamycin/neomycin genes.

TIA

Not surprising at all...false positives are common in cloning.  Even if you got no colonies on the control plates, having just one colony on a plate is very suspicious, true resistance will give lots of colonies always.

Kanamycin and geneticin are NOT the same thing.  Kanamycin resistance does not confer geneticin resistance.  All you did was waste your time and reagents plating something that doesn't work.

Do not trust the colony!

[leelee]

There IS a gene that confers resistance to both kanamycin AND geneticin- the neo resistance gene.
(and in fact, a quick google search located another, novel gene with the same dual resistance "A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species" Agaphonov et al, 2009, http://www3.intersci...4191/HTMLSTART)

It was my understanding that you should use Kn as the selection antibiotic in bacteria, and G418 in eukaryotic cells.