Plasmid size limit for transformation

Ed Schwartz edward=schwartz at rz.hu-berlin.de
Sun Oct 19 09:14:28 EST 1997


Torben ¯sterlund wrote:
> 
> I am about to make a construct that will be approx. 21 kb (including the
> 2.9 kb of the vector, pBluescript).
> My question is: will it be possible to transform E. Coli with this
> (monster) plasmid  by electroporation?. Should I adopt some special
> precautions???
>  Thanks for your tips!
> 
> Juan A. Contreras
> Lund University

I routinely make plasmids in the size range 23-26 kb and establish
them in standard E. coli strains. Its a little bit tricky. The critical 
step is not transformation but ligation. You should take a look at the
classic paper by Dugaiczyk et al. (1975) or Section 1.6 
in Sambrook. I always use dephosphorylated
vector. As a rule, I lower the concentration of fragments in the
ligation reaction and transform a larger volume of it. I usually
incubate longer (2-3h) before plating on selective plates. The
good old CaCl2 method works, so electroporation should be even better.
Hanahan recommends deo strains for large plasmids - dont ask me
why.
Good luck,
Ed Schwartz

Institut fuer Biologie/Mikrobiologie
Humboldt-Universitaet Berlin
edward=schwartz at rz.hu-berlin.de



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