E. coli electroporation efficiencies - pBR322 v/s pUC ori

Aawara Chowdhury aawara at FEMA-trailer.org
Sun Jun 25 15:57:34 EST 2006


Hi David,

Thanks for your response.  Actually, all the plasmids you've used have
high copy origins (in the sense that none of them have the "rop" gene).
All of them (including pUC) are essentially rop-deleted derivatives of
the pMB1 origin that is in pBR322.

Your experience does indicate to me that the relationship between size
and electroporation efficiency is not linear (even in the 2.5 kb - 12.5 kb
range).

Thanks,
AC

In <mailman.77.1151256667.23079.methods at net.bio.net>,
 Haviland, David L <David.L.Haviland at uth.tmc.edu> wrote:

> AC:
>
> I've found differences as well in transformation effeciency, also based on size.   But at the same time I've not rechecked the origin of replication of the respective vectors. 
>
> Currently, TOP10F's, and SURE cells have been our bugs of choice so that is where my current experience is.
>
> By electroporation, we have easily obtained 10^9 cfu/ug of pUC19 with both cells.   But as we know, this is also a small vector.   After knowing this, I then tried a nubmer of vectors we had in the lab.   pBluescript at 3kb rendered high 10^8 cfu/ug.  pCDNA3 at 5.3kb was in the low 10^8 cfu/ug.  With a 7 kb insert, for a total of 12.3 kb, we had plummeted to the low 10^7 cfu/ug.   The only other thing I had was a cosmid, checking in around 47kb (7kb for pTCF vector and about 40kb for the insert) the effiency plummeted to the mid 10^4 cfu/ug.
>
> I could be wrong but if memory serves most of these are using the old pBR322 backbone with its ORI of replication.
>
> So by comparison, I think we have similar results with different sized vectors.
>
> David
>
>
>
> -----Original Message-----
> From: methods-bounces at oat.bio.indiana.edu on behalf of Aawara Chowdhury
> Sent: Sat 6/24/2006 4:02 PM
> To: methods at magpie.bio.indiana.edu
> Subject: E. coli electroporation efficiencies - pBR322 v/s pUC ori
>  
> 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.
>
> AC
>
> 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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