high efficiency transformation

[Merijn Salverda]

Hi all,

I'm currently attempting to get high efficiency transformation of a 4
kb plasmid into DH5alfa (E. coli) using electroporation. I'm aiming at
an efficiency of 10e8 to 10e9, but so far I got stuck at 10e7. My
plasmid has a tetracycline-marker, and I recently came across an
article that reports lower transformation frequencies when selection
is made for tetracycline ('The result is a transformation efficiency
comparable to that of chemical transformation' - Hengen 1995). Does
any of you have any experience with this? And does anyone have any
suggestions how to get higher yields anyhow? I've been thinking about
ordering ultra-competent cells (e.g. Electromax DH5alfaE), but as I
need to do shiploads of transformations, this would make it a rather
expensive matter...

Thanks in advance!

Merijn.


Merijn Salverda
Laboratory of Genetics
Wageningen University
Arboretumlaan 4
6703 BD Wageningen
the Netherlands