Dear William, 

I remember a similarly frustrating experience when I was first doing transformations.  I suspect that the optimal electroproration conditions might vary not only with each model of electroporator, but also with each individual instrument. 

My suggestion would be that before you try homologous recombination, try transforming some basic vector like actin promoter:: GFP.  Vary the electroporation conditions to get the largest number of colonies. This feels like a dumb experiment but I bet it will save you time in the long run. 

Good luck!

Dan Dickinson
University of North Carolina


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> On Dec 20, 2013, at 4:01 PM, "Swatson, William S. (MU-Student)" <[log in to unmask]> wrote:
> 
> Dear all,
> 
> I have been trying to perform targeted gene disruption by homologous recombination in AX4 cells using electroporation with no success. The conditions for electroporation differ in almost every protocol I have laid hands on. This is probably owing to the different apparatus being used. We use a BIO-RAD gene pulser with the following conditions; 1 kV, 3 uF and 200 ohms. We use 0.4 cm cuvettes. Specific questions are, what conditions have been used successfully using this type of apparatus? Is a resistor necessary? Is it better to perform a single pulse or an additional pulse? I welcome any suggestions. Thank you and happy holidays.
> 
> William S. Swatson
> Graduate Student
> Department of Biological Sciences
> University of Missouri, Columbia