E. coli electroporation efficiencies - pBR322 v/s pUC ori
aawara at FEMA-trailer.org
Sat Jun 24 16:02:40 EST 2006
Is transformation by electroporation affected into E. coli affected
by the replication origin on the plasmid?
We test our electrocompetent DH5alpha using a 7.7 kb plasmid, and
get efficiencies around 3x10e7/ug - 4x10e7/ug.
Adjusting for plasmid size, this translates into about 1x10e8/ug for
pUC18 or pUC19, which several commercial suppliers of competent cells
use as a standard. I'm curious why we never achieve the > 5x10e8
transformants per ug that commercial suppliers claim in their literature.
Does this have anything to do with the replication origin on the plasmid?
The plasmid we use to test efficiency has the pMB1 origin (which has
the rop gene, and is present at 10 - 30 copies/cell). I know that pUC
is at a much higher copy number (>300 copies/cell).
If copy number does affect the ability of a transformed cell to form
an antibiotic-resistant colony, then I can believe that our competent
bacteria are about as good as can be made.
P.S. Note - we have made other strains of E. coli competent to
significantly different degrees (easily exceeding 1x10e9/ug for MC1061,
or being as low as 1x10e7/ug for STBL2).
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