double transformation in e.coli

[deespike]

HI all

I have been trying to figure out is it possible for 2 different vectors having the same origin of replication but different antibiotic selection gene (ampR and KanR) and different in copy numbers (one being High copy number and the other having low copy number) replicate in the same host or stay as double transformants?


Plz give some suggestions.

[SebaQ]

There are strains of E. coli harbouring the pLysS plasmid that are further transformable. This competent cells are comercial, so you can get your own from many manufacturers.

If you transform this competent cells, you will have a clone that's resistant to Cloramphenicol and another antibiotic.

If you're asking whether or not you can transform with two vectors at the same time, I think it'd be hard, but not impossible. I've not done it myself.

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[rc_labman]

Yes, double transformations are possible. Use electroporation rather than chemical transformation as chemical does not have high enough efficiency. I commonly use 2ul of a 1:10 dilution of each mini-prepped plasmid with 50ul of electro comp cells. Transform as usual. Recover in 1 ml SOC for 1hr, plate 100-250 ul of the recovery mix with appropriate antibiotics.

However, if memory serves, the plasmids need different origins of replication even if the antibiotics are different. I could be wrong on that though.

[Kneeanderthal]

You can still use chemical transformation, just use a higher concentration of your plasmids (5 uL undiluted prep of each plasmid, 25 uL comp cells). Additionally I like recovering in 300 uL and then plating 20 uL, 100 uL and 200 uL.