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High conc. CaCl2 inhibits electroporation??


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3 replies to this topic

#1 Curtis

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Posted 14 March 2009 - 07:39 AM

I was trying to do electroporation after addition of pEGFP.N2 to my TOP10 cells but the electroporator gives error all the time. then a friend of mine told me perhaps it is because of high concentration of CaCl2 in my TOP10 that the machine gives error. he said he also had the same problem and the company technicians told him he needs to reduce the CaCl2 concentration!!! why is that?...don't tell me I need to re-grow cells :(

#2 phage434

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Posted 14 March 2009 - 05:48 PM

Cells prepared for electroporation are washed in sterile water, and if frozen, resuspended in 10% sterile glycerol. There should be no CaCl2 anywhere near them. Perhaps you are confusing competent cell preparation for chemical tranformation, which does use CaCl2 (although alone, in the good protocols).

#3 Curtis

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Posted 15 March 2009 - 12:36 AM

ow....so you're saying never use cells in CaCl2 for electroporation?

always go for freeze-thaw? or chemical transformation if there is CaCl2?

thank you

#4 HomeBrew

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Posted 15 March 2009 - 04:51 AM

The time constant is determined by the field strength and the resistance of the sample. The resistance of the sample will be dramatically decreased in the presence of ionic salts, and thus salts in your sample should be avoided or minimized. Too much salt will cause a too small time constant, and may induce arching.

I think you're confusing chemically competent cells with electrocompetent cells -- each procedure relies on different methods for cell preparation.




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