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2 plasmids in one competent cell - is this possible? - Cloning query (Dec/20/2006 )

Is it ever possible for a chemically competent cell (such as DH10B) to take in more than one plasmid? If so, under what circumstances?


I dont know what would u do that, but anyway I suppose it would be easy to get double transformants in a plate with 2 antibiotics (considering different selection markers for each plasmid)

QUOTE (sfc @ Dec 20 2006, 03:04 PM)
Is it ever possible for a chemically competent cell (such as DH10B) to take in more than one plasmid? If so, under what circumstances?


i don't think electroporation can deliver 2plasmids in same cell in one time.
But first selection then electroporation of 2nd with different ab hould do the job.
Btw, considering mutliple copy plasmids, the bacteria can manage many plasmid ber cell. So i think it won't care if they are same or different


i think it's possible even it's chance ! rolleyes.gif


it's possible.(at least with chemical competent bacteria)
It's not rare that you have one plasmid that control the repression of a gene, and an other plasmid that contain the gene and control the expression of the gene.
First transform with one plasmid, select with antibiotics, make them competent, transform with second plasmid and select with both antibiotics.


Most likely a rare event, but not impossible to happen.

For mammalian cells, people have co-transfected 3 plasmids and selected with 2 different antibiotics, screened and sorted by FACS and got cells expressing 3 proteins of interest in the end. So why not for bacteria?

Regarding the mechanism of chemical transformation, if you have 2 plasmids, I don't see why an e. coli could not get a plasmid in on one side and another on the opposite site. I would only try this with purified plasmids thought, not with ligation mixtures.


As far as I know, it is possible to co-trasform bacterial cells with 2 plasmids. I remember something about the replication origins, that they have to be different. Could someone confirm?

It is also useful if the two plasmids have 2 different resistances, so they can be selected in a medium that contains both antibiotics. You'll probably have very few double transformants though...


what about transforming 2 plasmids into one competent cell at one time!! unsure.gif



I think it's very rare...
If it's frequent, people working on cDNA libraries will have a big headache, esp. those doing mass sequencing of these libraries. Imagine having regular event of overlaps of sequence due to 2 plasmids in a cell, when doing sequencing.

Anyway, I think fred_33's approach might work, if the cell likes both plasmids and there's no dilution of one plasmid over the other.

-I love MSGs!-

yes it is possible.
for example, there are the M15 bacteria, already transformed with a plasmid containing a repressor.
then you can transform them again, with a second plasmid containing your gene of interest whose expression will be regulated by the first plasmid expressing the repressor.

have a look on QIAexpressionist from qiagen.

of course, you need two antibiotics for selection.