Electroporation of HMW DNA??? ;-)

[Paul N Hengen]

In bionet.molbio.methds-reagnts, Dima Klenchin wrote:

> Spiros Servos <s.servos at ic.ac.uk> wrote:
> I want to electroporate a series of HMW (ca. 10Kb) recombinant plasmids 
> into E. coli. Does anyone have any advice on how I should best set about 
> doing this?

Well, 10 kb is hardly a high molecular weight plasmid in my book...but anyway...

| Check recent NAR (no earlier than Jul), "Methods section". There was a paper on
| just exactly that. In brief, 2 mm cuvettes, 2.5 kV, 40 ul, 200 Ohm. 

I just tried the Bio-Rad Gene Pulser as a demo using these conditions with DH5
alpha. Granted the cells were prepared by the method of Tang1994, by collecting
at OD600 of 0.94 - 0.95, and I used 20 ng of pBR322 per 40 ul zapped in the 0.2
cm gapped cuvette, I got 6-fold higher efficiency of transformation with a
simple heat shock for 2 minutes as compared to electroporation for around 4
milliseconds, 2.5 kV, 200 Ohm. I wasn't impressed!

@article{Tang1994,
author = "X. Tang
     and Y. Nakata
     and H.-O. Li
     and M. Zhang
     and H. Gao
     and A. Fujita
     and O. Sakatsume
     and T. Ohta
     and K. Yokoyama",
title = "The optimization of preparations of competent
cells for transformation of {{\em E. coli}}",
journal = "Nucl. Acids Res.",
volume = "22",
number = "14",
pages = "2857-2858",
comment = "1x10^8 transformants per ug DNA at O.D. 0.94",
year = "1994"}

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