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Electroporation query


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#1 Inbox

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Posted 22 March 2014 - 03:05 AM

Hi,

If voltage is 1.8kV and time of current passing is 3.50ms. and still electroporation did not give transformed coloines can we say that electroporation is poor.  Thanks.


Edited by Inbox, 22 March 2014 - 03:05 AM.


#2 phage434

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Posted 22 March 2014 - 07:49 AM

No, this does not tell enough. If you have no experience with the cells, then you need to vary the voltage (200-300 volt steps) to test optimal conditions. Are you allowing time for cells to recover and express resistance genes by growth on non-selective medium after electroporation? What is the gap in your cuvette? Thbis can make a very large difference, since what really counts is the field, measured in volts/cm. Too high a voltage will kill all your cells, too low will not transform your cells. You can test the killing efficiency by serial dilution and growth on non-selective medium. I've also read (but not yet tested) the idea that you can measure ATP concentration in the supernatent after electroporation as a way of detecting cell pore formation. People do this with a luminescent enzymatic ATP assay.



#3 Inbox

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Posted 23 March 2014 - 09:21 AM

Yes I am following 1 hr incubation of electroporated bacteria in LB media after electroporation. gap in cuvette is 0.1cm. 



#4 phage434

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Posted 23 March 2014 - 12:58 PM

That voltage sounds quite high for a 1mm cuvette. I'm surprised you don't see arcing of the cuvette (a loud pop). I would definitely try some lower voltages, or some cell viability assays.



#5 Inbox

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Posted 24 March 2014 - 12:48 AM

No I am not getting Arching. Do you think I should still go for lower voltage?



#6 phage434

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Posted 24 March 2014 - 08:52 AM

Yes, I do. What is the organism? Cell size can be a major determiner of the correct field strength.



#7 Inbox

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Posted 24 March 2014 - 10:59 AM

E.coli. Could cell be dying even if there is no arching?


Edited by Inbox, 24 March 2014 - 11:25 AM.


#8 phage434

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Posted 24 March 2014 - 11:51 AM

Yes. I would be doing a parallel electroporation of a known good plasmid at low concentration (10 pg) to test transformation efficiency.






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