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Distilled water for Electrocompetent Cell Preparation


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#1 azim58

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Posted 27 April 2011 - 08:50 AM

I am wondering why we should use pure distilled water to prepare electrocompetent cells. I would think that distilled water would lyse or harm the cell due to differences in osmotic pressure. People never use distilled water in cell culture.

I have also read that some people use 1 mM HEPES buffer pH 7 to prepare electrocompetent cells as well. However, I have also heard that it makes no difference to the final transformation efficiency whether one uses distilled water or HEPES.

Is the distilled water okay because everything is kept so cold or something?

#2 bob1

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Posted 28 April 2011 - 08:39 PM

Distilled water (or ultrafiltered and ion exchanged, such as MilliQ) is used to make most solutions in the lab... I don't think the water will be used by itself for any other part of the proceedure.

#3 leelee

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Posted 01 May 2011 - 10:16 PM

"I don't think the water will be used by itself", it actually is, bob1, for washing the bacteria. Every protocol that I have ever seen or used for preparation of electrocompetent bacteria uses sterile water for at least two of the wash steps.
It works just fine, don't worry :)

Edited by leelee, 01 May 2011 - 10:16 PM.


#4 phage434

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Posted 02 May 2011 - 03:56 AM

It works fine for E. coli, but not for other species that are osmotically sensitive. These species often use an electroporation buffer of 1 mM HEPES, pH 7.5 plus 280 mM sucrose, chilled.

#5 azim58

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Posted 05 May 2011 - 01:54 PM

It works fine for E. coli, but not for other species that are osmotically sensitive. These species often use an electroporation buffer of 1 mM HEPES, pH 7.5 plus 280 mM sucrose, chilled.



Hmmm. . .do you think there would be any benefit to providing the bacteria with a nice pH as well. Perhaps the electroporation efficiency would be a little higher? I guess this is nothing to fret about since just about everyone uses water with e. coli anyway.


I should also add that using HEPES instead of water seems to decrease the time constant when I electroporate, which may be a bad thing. Perhaps I need to change the voltage I electroporate with when I use HEPES. Since I don't know enough about this, I may be better off using the standard distilled water method. I would like to get the highest electroporation efficiency possible though since I am making a cDNA library.

Edited by azim58, 05 May 2011 - 02:01 PM.


#6 phage434

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Posted 05 May 2011 - 05:14 PM

I don't know how important the pH of the buffer is. The pulse length is important, but can be controlled by adjusting the capacitance of the gene pulser.

#7 azim58

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Posted 05 May 2011 - 08:31 PM

Thanks for the information "phage434".




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