transformation problem - (Sep/11/2006 )

I have tried electroporating my plasmid into the SURE electrocompetent cells thrice but am not successful. I used the 0.1cm gap cuvettes and the parameters as described, I chilled my cuvettes and microcentrifuge tubes and my electroporator. All my conditions are very optimal. To ensure that my cells were still alive when I plated them on my selection plates, I plated them on kanamycin plates too. The next day, I observed a lawn on my kanamycin plates. This means that my cells are still alive but doesn't seem to accept my plasmids. I transformed with 2ul of 50pg/ul (100pg) into 40ul of cells and 1ul of 10pg/ul (10pg) into 40ul of cells (this is as described in the protocol and I should be expecting some colonies). Any idea why I didn't get any colonies?

How interesting. My lab uses the same setup but I have never read the user manual.
We use 1.8kV. I seem to get more cells compared to 1.7kV along with more arcing problems too. (DNA has to be really really desalted and used at the right concentration. Or else Zeus sends a lightning bolt at your competent cells....zzap!)

But I don't think that is the problem. Has a gel been run on the ligation mix before electroporation?
It is to make sure
1- there is DNA in in the mix.
2- the DNA has actually ligated, but the appearance of DNA bands of higher molecular weight.

How interesting. My lab uses the same setup but I have never read the user manual.
We use 1.8kV. I seem to get more cells compared to 1.7kV along with more arcing problems too. (DNA has to be really really desalted and used at the right concentration. Or else Zeus sends a lightning bolt at your competent cells....zzap!)

But I don't think that is the problem. Has a gel been run on the ligation mix before electroporation?
It is to make sure
1- there is DNA in in the mix.
2- the DNA has actually ligated, but the appearance of DNA bands of higher molecular weight.

Okay,
But it remains, is there DNA being transformed into the cells?

Also are the plasmid selection markers right. It should be checked in this situation. It might be a similar problem to vetticus3, where somebody forgot to tell him his plamid is not AmpR (but maybe his cells are) but the plasmid is really (Kan/Neo)

Something to check out.